CN104284978B - The generation of class rotaviral particles in plant - Google Patents

Abstract

The invention provides the method that viruslike particle (VLP) is produced in plant.Methods described includes being introduced to the first nucleic acid in the part of the plant or the plant.First nucleic acid is included in the first control region of nucleotide sequence that is active in the plant and being operatively connectable to encode one or more rotavirus structural protein matter (such as, but not limited to rotavirus protein VP2).The nucleotide sequence can also include one or more amplification element and/or compartment targeting sequence.Second nucleic acid can be introduced in the part of the plant or the plant.Second nucleic acid is included in the second control region of nucleotide sequence that is active in the plant and being operatively connectable to encode one or more rotavirus structural protein matter (such as, but not limited to rotavirus protein VP6).It is optionally possible to the 3rd nucleic acid and/or the 4th nucleic acid are introduced in the part of the plant or the plant.3rd nucleic acid is included in the 3rd control region of nucleotide sequence that is active in the plant and being operatively connectable to encode one or more rotavirus structural protein matter (such as, but not limited to rotavirus protein VP4).4th nucleic acid is included in the 4th control region of nucleotide sequence that is active in the plant and being operatively connectable to encode one or more rotavirus structural protein matter (such as, but not limited to rotavirus protein VP7).The part of the plant or the plant is cultivated under conditions of the expression of nucleic acid is allowed, thereby produces RLP.

Description

The generation of class rotaviral particles in plant

Technical field

The present invention relates to rotavirus structural protein matter is produced in plant.More particularly it relates in plant It is middle to produce the viruslike particle for including rotavirus structural protein matter.

Background technology

Rotavirus infection is global problem, the main children for influenceing less than five years old.It causes serious enterogastritis, And cause death in the worst case.

Rotavirus is the member of Reoviridae (Reoviridae) viral (rotavirus), and it infects stomach system System and respiratory tract.The title, which comes to work as, passes through negative phase contrast electron microscope (negative contrast electron Colyliform outward appearance (Fig. 1 a of virion when microscopy) observing；Prior art).Rotavirus is generally spherical, and because Shell and inner casing or their bivalve capsid structure and gain the name.Respectively, a diameter of about 70nm of outer capsid, inner capsid are a diameter of about 55nm.The bivalve capsid of rotavirus surrounds the core for including internal protein shell and genome.The genome of rotavirus is by compiling The double stranded RNA fragment composition of at least 11 rotavirus proteins of code.

DsRNA encodes six structural protein matter (VP) and six nonstructural proteins (NSP) (Fig. 1 c；Existing skill Art).Structural protein matter includes VP1, VP2, VP3, VP4, VP6 and VP7 (Fig. 1 b；Prior art).Three concentric layers lead to respectively The assembling for crossing VP2, VP6 and VP7 is formed, and wherein VP4 is formed " furcella (spike) " on virus structure surface.NSPs be by The intracellular synthesis of infection, and work in some of replicative cycle or with some in host protein mutual Act on so as to influence morbidity or to the immune response of infection (Greenberg and Estes, 2009).

VP2 is 102kDa protein, and is the protein that content is most abundant in virus core.It forms innermost layer Structural protein matter layer, and component for virus core and the correct assembling of transcriptase provide skeleton (Lawton, 2000). VP1 is 125kDa maximum virus protein, and it is acted on as the RNA dependences polymerase of rotavirus, produces core Replicative intermediate, and be combined in its icosahedron apex with VP2 (Varani and Allain, 2002；Vende etc. People, 2002).VP3 (98kDa protein) is also directly combined with viral genome, adds 5 ' end cap configurations so as to play To the effect of viral mRNA mRNA capping enzymes (capping enzyme).VP1 is formed together with VP3 is attached to VP2 shell layers The 5 times of summits in outside compound (Angel, 2007).VP6 is 42kDa protein, and it forms the middle level shell of virus core, It is major capsid protein, and account in the gross protein material of virion more than 50% (Gonz á lez et al., 2004； Estes, 1996).Necessary to it is genetic transcription, and can be by the way that VP1 is anchored into VP2 and in colyliform disease in the core Worked in malicious RNA encapsulation, such as the institute in another member blue tongue rims (bluetougue virus) of Reoviridae As it was observed that.It also determines that rotavirus is divided into the classification of five groups (A to E), and wherein A groups most often infect the mankind (Palombo, 1999).VP6 in rotavirus A groups has at least four subgroups (SG)：SG I, SG II, SG (I+II) and SG Non- (I+II), this depends on the presence or absence of SG specificity epitopes.B groups lack the shared antigen of A groups, but it is also known that meeting with C groups Infection people, and D groups only infection animal, for example, chicken and milk cow (Thongprachum, 2010).

Two kinds of outside capsid protein VP7, i.e. the protease sensitive VP4 (P) of 37kDa glycoprotein (G) and 87kDa are defined The serotype of virus.The induction neutralizing antibody reaction of both protein simultaneously thus be used to rotavirus serotype being categorized as Double naming systems, this depend on G-P antigen combinations (for example, G1P [8] or G2P [4]) (Sanchez-Padilla et al., 2009).For VP4 protein dimerizations so as to form 60 furcellas on virus coat, the furcella directly participates in host cell entrance Starting stage.Spike protein includes cleavage site at amino acid (aa) position 248.Once infection, it is by protease pancreas egg White cleavage, so as to produce VP5 (529aa, 60kDa) and VP8 (246aa, 28kDa) (Denisova et al., 1999).The process Enhance the infectivity (the cell attachment and intrusion of host cell) of virus and stabilize furcella structure (Glass, 2006). VP7 glycoprotein forms the third layer or outer layer of virus.At present, 27G and 35P genotype are known (Greenberg and Estes, 2009).VP4 and VP7 be participate in the major antigen that virus neutralizes and be vaccine development important target (Dennehy, 2007)。

In infected mammalian cell, the form generation of rotavirus experience unique forms is to form complete three Layer VP2/6/4/7 virions (Lopez et al., 2005).Three layers of capsid are highly stable compounds, can excrement-mouth Propagate and by Viral delivery to small intestine, herein differentiation enterocyte of its infection close to the nondividing of fluff tip (Greenberg and Estes, 2009).First, complete virus is attached by 60 VP4 dimer furcellas on virus surface On the acceptor independently of sialic acid (Lundgren and Svensson, 2001).60 VP4 dimerization on virus surface Body furcella allows virus to be attached to these cell receptors.For VP4 to cutting sensitivity by the proteolysis of trypsase, this causes structure As change, the change is exposed for a series of extra attachment position interacted with coreceptors, on glycoprotein surface Point.

However, multistep is adhered to and not yet had a clear understanding of into process, but virus is passed through host cell membrane.VP7 Outside capsid (also assisted in and entered process) is removed in this process, and double layer particle (DLP) is delivered in vesica Cytoplasm (Fig. 2；Prior art).DLP departs from from vesica and enters the cytoplasmic inclusion (cytoplasmic of non-film combination Inclusions in).Started by VP1 genome early transcription in particle, so that dsRNA is never exposed to cell Matter.Rna replicon is formed in the cytoplasmic inclusion of these non-film combinations with core and occurred.Then, initial stage (+) RNA is transported Enter cytoplasm and as the template of virus protein synthesis.VP4 is produced in cytosol and is transported to rough surfaced endoplasmic reticulum (RER) (RER), and VP7 is secreted into RER.VP2 and VP6 is produced and assembled in the cytosol of virion, and then sprouts (bud) Enter RER compartments, obtain in this process instantaneous membrane envelope (Lopez et al., 2005；Tian et al., 1996).In RER, taking turns Under shape viral glycoprotein NSP4 key participation, the instantaneous membrane envelope of virion is removed and by VP4 and VP7 protein lists Body replacement (Tian et al., 1996；Lopez et al., 2005；Gonzalez et al., 2000).NSP4 is in ER films as intracellular Acceptor plays a role, and combine it is new caused by subviral particle, and be also possible to combine spike protein VP4 (Tian et al., 1996).NSP4 is also poisonous to people, and is the causative agent of diarrhoea.Then, complete, ripe particle is by golgiosome from RER In be transferred to plasma membrane and secreted (Lopez et al., 2005).

A variety of different means have been used to produce suitable protection crowd to keep out the wheel of the rotavirus of various serotype Shape viral vaccine.These means include a variety of Jennerian means, using live attenuated virus, use viruslike particle, nucleic acid epidemic disease Seedling and the viral subunit as immunogene.There are two kinds of available Oral vaccines in the market, however, in some development China Family is relatively low due to Strain change and the presence of other pathogen, these vaccine potencies.

United States Patent (USP) No.4,624,850,4,636,385,4,704,275,4,751,080,4,927,628,5,474, 773 and 5,695,767 every describe a variety of Rotavirus Vaccines and/or the method for preparing them.What the group membership shared is total to The same sex is that each in these vaccines all relies on the uses of whole virus particles to produce final Rotavirus Vaccine.Consider To the long-term needs of effective polyvaccine, it is evident that the operative body on solving demand to such vaccine only part into Work(.

Different from traditional vaccine production method, the progress of biology field has allowed to express each rotavirus protein. Crawford et al. (J Virol.1994September；68(9):5945-5952) by encode major capsid protein VP2, VP4, VP6 and VP7 are cloned into baculovirus expression system, and every kind of protein is expressed in insect cell.Rotavirus master The coexpression of the various combination of structural protein matter is wanted to result in stable viruslike particle (VLP).VP2 and VP6 list Solely coexpression or cause to generate VP2/6VLP or VP2/4/6VLP with VP4 coexpression, they are with double-deck rotaviral particles It is similar.VP2, VP6 and VP7 coexpression generate three layers of VP2/6/7VLP or VP2/4/ (in the case of with or without VP4) 6/7VLP, they are similar to natural infection rotaviral particles.Each VLP maintains the structural and functional feature of natural particulates On (as determined by by particle electron microscope observation), VP4 and VP7 the presence of non-neutralizing epitope and neutralizing epitope and VP2/4/6/7VLP Hemagglutination activity.

Vaccine candidate object caused by viruslike particle from different proteins composition is had shown that as subunit vaccine Potentiality.O ' Neal et al. (" Rotavirus Virus-like Particles Administered Mucosally Induce Protective Immunity,"J.Virology,71(11):8707-8717 (1997)) show and work as and will contain VP2's and VP6 When being applied to mouse in the case of adding and not adding cholera toxin, they are exempting from VLP or VLP containing VP2, VP6 and VP7 Induction of protective immunity in epidemic disease mouse, and protected when each VLP is applied together with cholera toxin (CT) more effective.

Class core granule (CLP) is additionally operable to immune milk cow, Fernandez et al. (" Passive Immunity to VLP Bovine Rotavirus in Newborn Calves Fed Colostrum Supplements From Cows Immunized with Recombinant SA11rotavirus core-like particle(CLP)or virus-like particle(VLP)vaccines,"Vaccine,16(5):507-516(1998)).In this study, have studied CLPs with VLPs produces the ability of passive immunity.The seminar summarizes：VLP is more more effective than CLP when inducing passive immunity.

Plant is just being increasingly used for the large-scale production of recombinant protein.For example, the disclosures of US 2003/0175303 Recombinant rotavirus structural protein matter VP6, VP2, VP4 or VP7 expression in tomato plant through stable conversion.

Saldana et al. uses cauliflower mosaic virus (CaMV) 35S promoter and recombinational agrobacterium (A.tumefaciens) VP2 and VP6 (Saldana et al., 2006) are expressed in the cytoplasm of tomato plant.Electron microscopic Mirror research shows that least a portion of particle has been assembled in 2/6VLP.Protective immune response, and this are detected in mouse Caused by being probably to a certain extent unassembled VP.Each protein, which has been shown in mouse, causes immune response, such as in VP8 With such (Zhou et al., 2010) in the case of VP6.

Matsumura et al. (2002) first reported the expression of bovine rota A VP6 in transgenic potato plant And assembling.In their research, they have used the transgenosis regulated and controled by cauliflower mosaic virus (CaMV) 35S promoter Potato plant and the recombinational agrobacterium (Agrobacterium tumefaciens) for carrying VP6 genes.By protein table Reach, purify and carry out immune Research.Immune response in adult mice shows in serum VP6 antibody be present.However, they do not show The sign of assembled VP6 protein is shown.It is probably that simple monomer or tripolymer (may cause immune answer in mouse Answer).The work of another group is shown using Potyvirus X (PVX) carriers at tobacco (Nicotiana benthamiana) Middle VP6 assembling (O ' Brien et al., 2000).When VP6 albumen is expressed in plant, find it only to be fused to PVX shaft-like Assembled during protein (PVX protein rods).Once cutting, VP6 is assembled into icosahedron VLP, such as Marusic People (2001) is such what is observed in the similar research to HIV-PVX.The result may imply that rotavirus protein can The extra factor or reinforcing can be needed to form VLP.

Due to needing both synthesis and assembling to one or more recombinant proteins, therefore VLP production is that have The work of challenge.For rotavirus, (it is RNA virus, has what is formed by 1860 monomers of four kinds of different proteins Capsid) VLP for just so.Production for VLP, expression is required with assembling while two to three recombinant proteins 's.These include VP2 (internal layer) of 120 molecules, 780 molecules VP6 (intermediate layer) and 780 molecules glycoprotein VP7 (outer layer), ultimately form double-deck or three layers of particle.In addition, table while most of VLP production some recombinant proteins of needs Up to assembling, in the case of class rotaviral particles (RLP) for, this needs occurs in single host cell.

Nearest research is shown to be mediated using beet black scorch virus (Beet black scorch virus, BBSV) Expression system in the red goosefoot (Chenopodium amaranticolor) people colyliform disease of the successful expression through codon optimization Malicious VP6.The protein is engineered to the sub of BBSV coat protein ORFs.Use the VP6 eggs based on plant Peroral immunity is carried out to female BAl BIc/c mouse in vain, induction of high-titer anti-VP6 mucous membranes IgA and serum IgG (Zhou et al., 2010).However, the seminar does not refer to that VP6 albumen is assembled into VLP or particle.

Rotavirus VP 7 is also successfully expressed into tobacco plant, and it is shown in mouse and maintained wherein With property immune response (Yu and Langridge, 2001).Shown using transgenic potato plant to express VP7 another research Show that VP7 genes are stable more than 50 generations in inverted plant.VP7 albumen from the 50th generation is in adult mice induction of guarantor Shield property both antibody and neutrality antibody (Li et al., 2006).

Yang et al. (Yang Y M, Li X, Yang H et al., 2011) is by A RV groups (P [8] G1) three rotavirus Capsid protein matter VP2, VP6 and VP7 are co-expressed into tobacco plant, and have studied these protein expressions it is horizontal and The formation of class rotaviral particles and immunogenicity.VLP has been purified from transgenic tobacco plant and by electron microscope and Western blot method is analyzed.Yang et al. result shows VP2, VP6 and VP7 protein self assembly of plant origin Enter diameter 60-80nm 2/6 or 2/6/7 class rotaviral particles.

The content of the invention

The present invention relates to rotavirus structural protein matter is produced in plant.More specifically, the invention further relates to planting The viruslike particle for including rotavirus structural protein matter is produced in thing.

According to the present invention, there is provided produce the method (A) of class rotaviral particles (RLP), methods described bag in plant Include：

A) following first nucleic acid, the second nucleic acid and the 3rd nucleic acid are introduced to the plant, the part of plant or plant cell In, first nucleic acid is included in active in the plant and is operatively connectable to encode the first colyliform virus structural First control region of the first nucleotide sequence of protein, second nucleic acid are included in active in the plant and operated Property be connected to coding the second rotavirus structural protein matter the second nucleotide sequence the second control region, the 3rd core Acid is included in active in the plant and is operatively connectable to encode the 3rd of third round shape viral structural proteinses matter 3rd control region of nucleotide sequence,

B) portion of the plant, plant is cultivated under conditions of the first, second, and third nucleic acid transient expression is allowed Point or plant cell, thus produce RLP.

In addition, in step a), following 4th nucleic acid can be introduced to the plant, the part of plant or plant cell, 4th nucleic acid is included in active in plant and is operatively connectable to encode fourth round shape viral structural proteinses 4th control region of the tetranucleotide sequence of matter, and it is thin when cultivating the plant, the part of plant or plant in step b) The 4th nucleic acid is expressed described in during born of the same parents.

As described in above method (A), the first colyliform viral structural proteinses matter can be VP2, the second rotavirus knot Structure protein can be VP6, and third round shape viral structural proteinses matter can be VP4 or VP7.In addition, fourth round shape Viral structural proteinses matter can be VP7 or VP4.Protease can co-express in plant.

Present invention also offers the method (B) for producing class rotaviral particles (RLP), methods described includes：

A) following nucleic acid are introduced to plant, the part of plant or plant cell, the nucleic acid, which is included in plant, to be had Activity and it is operatively connectable to encode the first nucleotide sequence of one or more rotavirus structural protein matter Control region,

B) to cultivate the plant, the part of plant or plant under conditions of the first nucleic acid transient expression is allowed thin Born of the same parents, thus produce RLP.

Method (B) as described above can also include：The second nucleic acid is introduced in step a), second nucleic acid is included in It is active and be operatively connectable to encode the second of one or more rotavirus structural protein matter in the plant Second control region of nucleotide sequence, also, expressed when cultivating the plant, the part of plant or plant cell in step b) Second nucleic acid.

Method (B) as described above can also include：The 3rd nucleic acid is introduced to the plant in step a), the described 3rd Nucleic acid is included in active in the plant and is operatively connectable to encode the structural egg of one or more rotavirus 3rd control region of the trinucleotide sequence of white matter, also, when the cultivation plant, the part of plant or plant in step b) The 3rd nucleic acid is expressed during cell.

In addition, in method (A) or (B), can be expressed in the plant, the part of plant or plant cell following other Nucleic acid, and wherein described other nucleic acid be included in the plant in it is active and be operatively connectable to encode silence The control region of the nucleotide sequence of repressor.

The use of nucleotide sequence codon can be adjusted to preferable people's codon use, the G/C content that improves or its Combination.

Rotavirus structural protein matter can include the natural or non-native signal peptide truncated.Non-natural signal peptide can To be protein disulfide isomerase signal (PDI) peptide.

First, second, third or tetranucleotide sequence or its combination operation can be connected to Cowpea Malicious (CPMV) control region.

Method (A) as described above or (B) can also comprise the following steps：

C) plant, the part of plant or plant cell are harvested, and

D) RLP is purified from the plant, the part of plant or plant cell.

During harvest or purification step in method (A) or (B), trypsase, trypsin-like protease can be used Enzyme, serine protease, chymotrypsin-like proteinase, subtilopeptidase A processing or cutting VP4, to produce VP5 and VP8.

RLP size can be in the range of 70-100nm, and can be purified in the presence of calcium.

The invention provides pass through RLP caused by method as described above (A) or (B).Caused RLP can be wrapped at least The structural protein matter of rotavirus containing VP4.Protease can be used, for example, trypsase, trypsin like proteases, silk ammonia Pepsin, chymotrypsin-like proteinase, subtilopeptidase A, VP4 is cut into VP5 and VP8.Protease can be in plant Interior coexpression adds during harvest, purifying or both.In addition, can be double by RLP caused by method (A) or (B) RLP and/or three layer of RLP of layer.

In addition, the invention provides nucleotide sequence.Encoding VP2 nucleotide sequence can include and such as SEQ ID NO： 13、SEQ ID NO：14 or SEQ ID NO：The homogeneity of nucleotide sequence 80% to 100% defined in 45.Encode VP6's Nucleotide sequence can include and such as SEQ ID NO：17、SEQ ID NO：18 or SEQ ID NO：Nucleotides sequence defined in 46 The homogeneity of row 80% to 100%.Encoding VP7 nucleotide sequence can include and such as SEQ ID NO：19、20、48、49、 52nd, the homogeneity of nucleotide sequence 80% to 100% defined in 53,54 or 57.Also, the nucleotide sequence for encoding VP4 can With comprising with SEQ ID NO：15th, the homogeneity of nucleotide sequence 80% to 100% defined in 16,47,50 or 51.In addition, VP2 can by comprising with SEQ ID NO：1 or SEQ ID NO：80% to 100% homogeneity of amino acid sequence defined in 25 Amino acid sequence encodes.VP6 can by comprising with SEQ ID NO：3 or SEQ ID NO：80% of amino acid sequence defined in 31 Amino acid sequence to 100% homogeneity encodes.VP7 can by comprising with SEQ ID NO：4th, amino defined in 39,43 or 59 The amino acid sequence coding of 80% to 100% homogeneity of acid sequence.VP4 can by comprising with SEQ ID NO：2 or SEQ ID NO：36th, the amino acid sequence coding of 80% to 100% homogeneity of amino acid sequence defined in 33.It is one or more Individual rotavirus structural protein matter can be VP2, VP4, VP6 and/or VP7.VP4 can be processed into VP5 and VP8.One Or more rotavirus structural protein matter can be selected from rotavirus strain G9P [6], rotavirus A WA strains, rotavirus A Vaccine USA/Rotarix-A41CB052A/1988/G1P1A [8] strains and rotavirus SA11 strain.

In method as described above (A), first, second or third nucleotide sequence or its combination can include it is following at this Active control region in plant, the control region are operatively connected to one or more cowpea mosaic virus (comovirus) enhancer, be connected to one or more amplification elements and be connected to coding colyliform virus structural egg The nucleotide sequence of white matter, and wherein can by encode replicase the 4th nucleic acid be introduced to the plant, plant part or Plant cell.

In method as described above (B), first, second, third or the 4th nucleotide sequence or its combination can include down Active control region in the plant is stated, the control region is operatively connected to one or more Cowpea Mosaics Viral (comovirus) enhancer, it is connected to one or more amplification elements and is connected to coding colyliform virus structure Property protein nucleotide sequence, and wherein the 5th nucleic acid that encode replicase can be introduced to the portion of the plant, plant Point or plant cell.

In addition, according to the present invention, there is provided produce the method (C) of class rotaviral particles (RLP), the side in plant Method includes：

A) following nucleic acid are introduced to the part of plant or plant, the nucleic acid is included in active in plant and grasped The control region for the nucleotide sequence for encoding one or more rotavirus structural protein matter is operatively connected to,

B) part of the plant, plant is cultivated under conditions of the first nucleic acid transient expression is allowed, is thus produced RLP。

Furthermore, it is possible to which the second nucleic acid to be introduced to the part of plant or plant, second nucleic acid is included in the plant The second nucleotides sequence that is active and being operatively connectable to encode one or more rotavirus structural protein matter Second control region of row, and wherein when cultivating the part of the plant or plant in step b), the second nucleic acid is expressed.

Furthermore, it is possible to which the 3rd nucleic acid to be introduced to the part of plant or plant, the 3rd nucleic acid is included in the plant Trinucleotide sequence that is active and being operatively connectable to encode one or more rotavirus structural protein matter 3rd control region of row, and wherein when cultivating the part of the plant or plant in step b), the 3rd nucleic acid is expressed.

Method (C) as described above can also include the step of harvest plant and extraction RLP.

One or more rotavirus structural protein matter of method (C) can be rotavirus protein VP2, VP4 Or VP6.Described first or second nucleotide sequence coded one or more rotavirus structural protein matter can be VP2 Or VP6.One or more rotavirus structural protein matter of trinucleotide sequential coding can be VP4.Can be by VP4 Cut to produce VP5 and VP8.

First, second or third nucleotide sequence can also be encoded, include or encoded and comprising one or one Compartment targeting sequence above and/or amplification element.One or more compartment targets sequence by one or more wheels Shape viral structural proteinses matter guides endoplasmic reticulum (ER), chloroplaset, plastid or the apoplast of plant cell into.Compartment targeting sequence can Encode apoplast signal peptide or plastid signal peptide.

Present invention also offers the method (D) for producing class rotaviral particles (RLP), methods described includes：

A) provide comprising following nucleic acid plant or plant part, the nucleic acid be included in plant in it is active and It is operatively connectable to encode the control region of the nucleotide sequence of one or more rotavirus structural protein matter,

B) plant, the part of plant or plant cell are cultivated under conditions of the nucleic acid transient expression is allowed, by This produces RLP.

In addition, the plant of method (D) or the part of plant can also include：

I) the second nucleic acid, it is included in active in plant and is operatively connectable to encode one or more wheels Second control region of the second nucleotide sequence of shape viral structural proteinses matter,

Ii) second and the 3rd nucleic acid, wherein second nucleic acid is included in active in plant and operatively connected The second control region of the second nucleotide sequence for encoding one or more rotavirus structural protein matter is connected to, and it is described 3rd nucleic acid is included in active in plant and is operatively connectable to encode one or more rotavirus structural 3rd control region of the trinucleotide sequence of protein,

Wherein when cultivating the part of plant or plant in step b), second nucleic acid or described second and the 3rd Nucleic acid is expressed.

One or more structural protein matter in method (D) can be rotavirus protein VP2, VP4 or VP6. Described first or second nucleotide sequence coded one or more rotavirus structural protein matter can be VP2 or VP6. One or more rotavirus structural protein matter of the trinucleotide sequential coding can be VP4.Egg can be used White enzyme, for example, trypsase, trypsin like proteases, serine protease, chymotrypsin-like proteinase, hay bacillus egg White enzyme, VP4 is cut into VP5 and VP8.Protease can co-express in plant, or add during harvest, purifying or both Enter.

The invention provides pass through method as described above (A), method (B), method (C), method (D) or combinations thereof Caused RLP.The RLP can include one or more rotavirus structural protein matter, and it is special that it can include plant Property N- glycan or the N- glycan through modification.

The present invention includes the composition of the RLP containing effective dose and pharmaceutical acceptable carrier, and the RLP is by such as institute just now Prepared by method (A), method (B), method (C), method (D) or the combinations thereof stated, for inducing immune response.

Present invention additionally comprises to the method for the immunity of rotavirus infection, methods described includes will for induction in subject RLP as just mentioned is applied to subject.Can by RLP oral, intracutaneous, intranasal, intramuscular, intraperitoneal, intravenously or subcutaneously It is applied to subject.

Present invention also offers vegetable material, the vegetable material include by method as described above (A), method (B), Method (C), method (D) or RLP caused by combinations thereof.It is sick to colyliform that vegetable material can be used for the induction in subject The immunity of poison infection.Vegetable material is also used as food supplement and is mixed.

In method as described above (method A, B, C or D), the part of plant or plant can also suppress with coding silence Another nucleotide sequence of son is applied together, or can also include another nucleotide sequence of coding silencing suppressor.

In addition, present invention also offers the method (E) that rotavirus structural protein matter is produced in plant, methods described Including：

A) following nucleic acid are introduced to the part of plant or plant, the nucleic acid is included in active in plant and grasped Operatively it is connected to the control region for the nucleotide sequence for encoding one or more rotavirus structural protein matter；

B) part of the plant or plant is cultivated under conditions of the nucleic acid transient expression is allowed, thus produces one Or more rotavirus structural protein matter.

Present invention does not mean that the whole features for describing the present invention.

Brief description of the drawings

The description below carried out by reference to accompanying drawing, these and other features of the invention will become apparent, wherein：

Fig. 1 shows rotavirus structure and the distribution of gene-protein matter.(A) transmission electron microscopy of rotaviral particles As a result (scale bar represents 100nm).(B) tissue of viral capsid proteins matter, it include it is internal, middle with it is outside.(C) according to big The small rotavirus dsRNA segments with function arrangement.DsRNA (D) can be separated by polyacrylamide gel electrophoresis.Pass through (D) the dsRNA segments in represent the protein in (C).Picture comes from：Crawford et al., 1997 (A), Swiss Institute of Bioinformatics, 2008 (B) and Greenberg and Estes, 2009 (D).

Fig. 2 shows that the cell of rotavirus enters and duplication.When rotavirus enters cell, VP4 and VP7 loses, So as to form double layer particle (DLP).DsRNA transcription starts, so as to cause VP2, VP4, VP6 and VP7 translation.In viral work Factory's (also referred to as viroplasm) is generated with the filial generation core for replicating enzymatic activity.The later stage turn occurs in these filial generation cores Record.Around viroplasm (virus factories), these cores are coated by VP6, so as to form budding and pass through endoplasm The prematurity DLP of nethike embrane, obtain the instantaneous adipose membrane that viral glycoprotein NSP4 and VP7 modifications are resident through ER；The particle of these coatings Also include VP4.When particle is towards during movement inside ER cisternas (cisternae), instantaneous adipose membrane and nonstructural proteins NSP4 loses Mistake, and virus surface proteins VP4 and VP7 is reset to form outermost virus protein layer, it is infectious so as to produce having for maturation Three layers of particle (referring to Swiss Institute of Bioinformatics (ViralZone)：viralzone.expasy. org/viralzone/all_by_species/107.html)。

Fig. 3 shows agrobacterium vector pTRAc, pTRAkc-rbcs1-cTP and pTRAkc-ERH.P35SS, have and repeat The CaMV 35S promoters of transcriptional enhancer；CHS, 5 ' non-translational regions of chalcone synthase (chalcone synthase)； PA35S, CaMV 35S polyadenylation signals；SAR, skeleton land (the scaffold attachment of tobacco Rb7 genes region)；The left margin and right margin that LB and RB, T-DNA are integrated；ColE1ori, the replication orgin of Escherichia coli (E.coli)； RK2ori, the replication orgin of Agrobacterium (Agrobacterium)；Bla, ampicillin/Carbenicillin resistance bla genes； LPH, the signal peptide sequence from mouse mAb24 heavy chains；His6,6 × His flag sequence；SEKDEL, ER- retention signal sequences； Rbcs1-cTP, the chloroplaset of the Rubisco little subunits gene (rbcS1) from potato (Solanum tuberosum) turn Transport peptide sequence；Npt II, kalamycin resistance npt II genes；Pnos and pAnos, nopaline synthase (nopaline Synthase) promoter of gene and polyadenylation signal (Maclean et al., 2007).

Fig. 4 shows the overview of rotavirus clone and infiltration (infiltration) flow.

Fig. 5 shows apoplast Protein Extraction flow.(A) plant cell and apoplast position are demonstrated.VP albumen Matter is expressed in cytosol, and targets apoplast (red arrow).(B)-and after the time tests, by plant leaf blade PBS Vacuum immersion (1), and be placed in perforation centrifugal column (2), then centrifuged in 2ml microcentrifugal tubes (Eppendorf) to receive Collect juice (3).

Fig. 6 shows the Western prints to the protein expression of rotavirus vp 6 in 7 days in leaves of plants cellular compartment Mark is analyzed.Use mouse anti-rotavirus VP6 antibody (1:5000) to be detected to film.(+) and (-) be illustrated respectively in or There is no the expression in the case of silencing suppressor.Red line represent VP6 protein it is multiple analysis samples in position (~ 40kDa).VP6 expression and extraction efficiency are optimal in cytoplasm.

Fig. 7 shows western blot, and which show planted at the 3rd day, in Ben Saimushi tobaccos (N.benthamiana) The respective expression of rotavirus protein through histidine mark in the cytoplasm of strain blade.The colyliform disease of+ve-bacterial expression Malicious VP2；M-molecular weight marker；VP-Rotavirus capsid protein matter.VP7 infiltrations cause blade (b) yellow.

Fig. 8 is shown in the 3rd day, targeting multiple Ben Saimushi tobaccos (N.benthamiana) leaves of plants cellular compartments Rotavirus vp 2 (a) and VP4 (b) expression.Use the chicken anti-rotavirus serum (1 for being directed to VP2 and VP4 respectively:2000) come Detect protein.CTp-chloroplaset；ER-endoplasmic reticulum；PTRAc-cytoplasm；A-apoplast；Negative control (- ve)-only use silence The plant of repressor infiltration；(a) VP2 of positive controls (+ve)-bacterial expression in, positive controls-bacterium in (b) The VP4 of expression；(- with+) it is with or without silencing suppressor；M-molecular weight marker.Arrow represents protein band of interest Position.

Fig. 9 shows the VP2/6/4 to being co-expressed in the cytoplasm of Ben Saimushi tobaccos (N.benthamiana) blade The 3rd day extract Western blot analysis.With chicken anti-rotavirus serum (anti-VP2 (1/5000) and anti-VP4 (1/ ) and the anti-VP6 antibody (1 of mouse 5000):5000) mixture detection protein.Recombinational agrobacterium is completed with silencing suppressor Infiltration.Negative control (- ve)-only with the whole plant of silencing suppressor infiltration；M-molecular weight marker.

Figure 10 shows the rotavirus egg that the 3rd day cytoplasm through uranyl acetate (uranyl acetate) dyeing extracts The electron microscope picture of white matter.(a) there is the negative proteins quality sample extract of silencing suppressor；(b) VP6 Protein Extractions Thing；(c) VP2/6 protein extracts and (d) VP2/6/4 protein extracts.Scale bar=200nm.The RLP's of all detections Diameter is between 70-100nm.(b) arrow in represents VP6 sheaths/pad (sheath/mat).(C) arrow in represents aRLP An example.All proteins are expressed in the case where silencing suppressor be present.All proteins are resisted with mouse VP6 antibody (1:2000) capture.

Figure 11 shows the VP2/6 and VP2/6/4 of coexpression sucrose gradient purified (a).Through sucrose gradient purified VP2/6 (b) and VP2/6/4 (c) Dot blot.Protein extract is loaded on saccharose gradient (10-60%) and exceeded the speed limit Centrifugation.By using the anti-VP6 antibody (1 of (b) mouse:And the anti-VP2 and VP4 serum (1 of (c) chicken 5000):5000) detection carrys out analysis level Point.

Figure 12 shows the Western blot analysis (a) to VP2/6 fraction, to fraction VP2/6 fraction 16 and 17 SDS-PAGE Coomassie-stained gels photos (b), and the Western blot analysis (C) to fraction 16 and 17.Printed in Western The anti-VP6 (1 of mouse is used in mark method (a):And the anti-VP2 serum (1 of chicken 5000):5000), and in (c) resist using only mouse VP6(1:5000).(a) with the VP4 of negative control (- ve)-bacterial expression in (c), negative control (- ve)-use in (b) is heavy Silent repressor infiltration and with sucrose gradient purified plant；Rough-unpurified VP2/6 extracts；(a) positive control in The VP2 of (+ve)-bacterial expression, (b) and the VP6 of the positive control (+ve) in (c)-plant expression；VP6-SF9-in SF9 insects The VP6 protein for the concentration known expressed in cell.Arrow represents discussed protein band.

Figure 13 shows the measure of total soluble protein in the fraction to the VP2/6 purified through sucrose density gradient. (a)-IgG standard curves, the absorbance readings of the fraction read under (b) -750nm.The point of concern：Fraction 16 to 19.

Figure 14 shows the analysis of sugar density gradient of the VP2/6/4 fractions to cytoplasm coexpression.Read in 750nm former Beginning absorbance readings, to verify the protein peak previously detected on VP2/6/4 Dot blot.

Figure 15 shows the transmission electron micrograph of the VP2/6 particles through sucrose density gradient purifying.(a) and Both (b) it is shown in the two kinds of different sections observed on copper mesh.Detected all RLP diameter 70-100nm it Between.With the anti-VP6 antibody (1 of mouse:2000) sample is captured.Scale bar represents 200nm.

Figure 16 a show amino acid sequence (the SEQ ID NO of rotavirus vp 2：And nucleotide sequence (SEQ ID 1) NO：13 and 14).Figure 16 b show amino acid sequence (the SEQ ID NO of rotavirus vp 4：And nucleotide sequence (SEQ ID 2) NO：15 and 16).Figure 16 c show amino acid sequence (the SEQ ID NO of rotavirus vp 6：And nucleotide sequence (SEQ ID 3) NO：17 and 18).Figure 16 d show amino acid sequence (the SEQ ID NO of rotavirus VP 7：And nucleotide sequence (SEQ ID 4) NO：19 and 20).

Figure 17 A show primer I F-WA_VP2 (opt) .s1+3c nucleotide sequence (SEQ ID NO：21).Figure 17 B show Primer I F-WA_VP2 (opt) .s1-4r nucleotide sequence (SEQ ID NO are shown：22).Figure 17 C show construct 1191 Schematic diagram.SacII and StuI restriction enzyme sites for plasmid linearization are annotated in schematic diagram.

Figure 18 shows construct 1191 from left t-DNA borders to the nucleotide sequence of right t-DNA borders (underscore) (SEQ ID NO：23).2X35S/CPMV-HT/NOS with plastocyanin-P19- plastocyanin silencing suppressor expression cassettes.

Figure 19 shows VP2 (opt) of the coding from rotavirus A WA strains nucleotide sequence (SEQ ID NO：45).

Figure 20 shows amino acid sequence (the SEQ ID NO of the VP2 from rotavirus A WA strains：25).

Figure 21 shows the schematic diagram of construct 1710.

Figure 22 A show the schematic diagram of construct 193.SacII and StuI restriction enzyme sites quilt for plasmid linearization Annotate on schematic diagram.Figure 22 B show nucleotide sequence (the SEQ ID NO of construct 193：26).Construct 193 with from The form on left t-DNA borders to right t-DNA borders is shown (underscore).2X35S/CPMV-HT/NOS with carry plastocyanin- The mode of P19- plastocyanin silencing suppressor expression cassettes enters BeYDV (m)+replicase amplification system.

Figure 23 shows nucleotide sequence (the SEQ ID NO of expression cassette 1710：27).Expression cassette 1710 is with from 2X35S The form of promoter to NOS terminator is shown.VP2 (opt) from rotavirus A WA strains is represented with underscore.

Figure 24 shows the schematic diagram of construct 1711.

Figure 25 A show primer I F-WA_VP6 (opt) .s1+3c nucleotide sequence (SEQ ID NO：28).Figure 25 B show Primer I F-WA_VP6 (opt) .s1-4r nucleotide sequence (SEQ ID NO are shown：29).Figure 25 c, which are shown from 2X35S, to be started Sub (the SEQ ID NO of expression cassette 1713 to NOS terminator：30).VP6 (opt) from rotavirus A WA strains is drawn below Line represents.Figure 25 d show VP6 (opt) of the coding from rotavirus A WA strains nucleotide sequence (SEQ ID NO：46).

Figure 26 shows amino acid sequence (the SEQ ID NO of the VP6 from rotavirus A WA strains：31).

Figure 27 shows the schematic diagram of construct 1713.

Figure 28 shows nucleotide sequence (the SEQ ID from 2X35S promoters to NOS terminator of expression cassette 1714 NO：32).VP6 (opt) from rotavirus A WA strains is represented with underscore.

Figure 29 shows the schematic diagram of construct 1714.

Figure 30 A show primer I F-Rtx_VP4 (opt) .s1+3c nucleotide sequence (SEQ ID NO：33).Figure 30 B Show primer I F-Rtx_VP4 (opt) .s1-4r nucleotide sequence (SEQ ID NO：34).

Figure 31 A show nucleotide sequence (the SEQ ID from 2X35S promoters to NOS terminator of expression cassette 1731 NO：35).VP4 (opt) from rotavirus A Rotarix strains is represented with underscore.Figure 31 B are shown from RVA/ The rotavirus A VP4 of Vaccine/USA/Rotarix-A41CB052A/1988/G1P1A [8] strain optimized code sequence Arrange (SEQ ID NO：47).Figure 31 C show the nucleotides sequence from 2X35S promoters to NOS terminator of expression cassette 1730 Arrange (SEQ ID NO：44).VP4 (opt) from rotavirus A Rotarix strains is represented with underscore.

Figure 32 shows amino acid sequence (the SEQ ID NO of the VP4 from rotavirus A Rotarix strains：36).

Figure 33 A show the schematic diagram of construct 1730.Figure 33 B show the schematic diagram of construct 1731.

Figure 34 A show primer I F-Rtx_VP7 (opt) .s1+3c nucleotide sequence (SEQ ID NO：37).Figure 34 B Show primer I F-Rtx_VP7 (opt) .s1-4r nucleotide sequence (SEQ ID NO：38).Figure 34 C are shown from 2X35S Promoter to the expression cassette 1733 of NOS terminator nucleotide sequence.From rotavirus A vaccines USA/Rotarix- The VP7 of A41CB052A/1988/G1P1A [8] strain represents (SEQ ID NO with underscore：24).Figure 34 D show that coding comes from The VP7 of rotavirus A vaccines USA/Rotarix-A41CB052A/1988/G1P1A [8] strain nucleotide sequence (SEQ ID NO：48).Figure 34 E show the colyliform from RVA/Vaccine/USA/Rotarix-A41CB052A/1988/G1P1A [8] strain Viral A VP7 optimized coded sequence (SEQ ID NO：54).

Figure 35 shows the VP7 from rotavirus A vaccines USA/Rotarix-A41CB052A/1988/G1P1A [8] strain Amino acid sequence (SEQ ID NO：39).

Figure 36 shows the schematic diagram of construct 1733.

Figure 37 shows primer I F-Rtx_VP7 (opt) .s2+4c nucleotide sequence (SEQ ID NO：40).

Figure 38 shows the schematic diagram of construct 1192.SacII and StuI restriction enzyme sites quilt for plasmid linearization Annotate on schematic diagram.

Figure 39 shows construct 1192 from the nucleotide sequence (underscore) on left t-DNA borders to right t-DNA borders (SEQ ID NO：41).2X35S/CPMV-HT/ with plastocyanin-P19- plastocyanin silencing suppressor expression cassettes PDISP/NOS is illustrated.

Figure 40 A show nucleotide sequence (the SEQ ID from 2X35S promoters to NOS terminator of expression cassette 1735 NO：42).PDISP/VP7 from rotavirus A vaccines USA/Rotarix-A41CB052A/1988/G1P1A [8] strain (opt) represented with underscore.Figure 40 B show that coding comes from rotavirus A vaccines USA/Rotarix-A41CB052A/1988/ The PDISP/VP7 (opt) of G1P1A [8] strain nucleotide sequence (SEQ ID NO：49).

Figure 41 is shown from rotavirus A vaccines USA/Rotarix-A41CB052A/1988/G1P1A [8] strain PDISP/VP7 amino acid sequence (SEQ ID NO：43).

Figure 42 shows the schematic diagram of construct 1735.

Figure 43 A show the rotavirus A from RVA/Simian-tc/ZAF/SA11-H96/1958/G3P5B [2] strain VP4 coded sequence (SEQ ID NO：50).Figure 43 B are shown from RVA/Simian-tc/ZAF/SA11-H96/1958/ The rotavirus AVP4 of G3P5B [2] strain optimized coded sequence (SEQ ID NO：51).Figure 43 C are shown from RVA/ The rotavirus A VP7 of Simian-tc/ZAF/SA11-H96/1958/G3P5B [2] strain coded sequence (SEQ ID NO： 52).Figure 43 D show the rotavirus A VP7 from RVA/Simian-tc/ZAF/SA11-H96/1958/G3P5B [2] strain Optimized coded sequence (SEQ ID NO：53).

Figure 44 A show primer I F-TrSP+Rtx_VP7 (opt) .s1+3c nucleotide sequence (SEQ ID NO：55). Figure 44 B show primer I F-Rtx_VP7 (opt) .s1-4r nucleotide sequence (SEQ ID NO：56).Figure 44 C, which are shown, to be come From the optimized of the rotavirus A VP7 of RVA/Vaccine/USA/Rotarix-A41CB052A/1988/G1P1A [8] strain Nucleotide sequence (the SEQ ID NO of coded sequence：57).Figure 44 D show expression cassette 1734 from 2X35S promoters to Nucleotide sequence (the SEQ ID NO of NOS terminator：58).From rotavirus A vaccines USA/Rotarix-A41CB052A/ The VP7 of 1988/G1P1A [8] strain is represented with underscore.Figure 44 E are shown from rotavirus A vaccines USA/Rotarix- The TrSp-VP7 of A41CB052A/1988/G1P1A [8] strain amino acid sequence (SEQ ID NO：59).Figure 44 F show structure The schematic diagram that body 1734.

Figure 45 is shown by Iodixanol (iodixanol) density gradient centrifugation to the class colyliform disease comprising VP2 and VP6 The purifying of malicious particle.Figure 45 A are that the coomassie carried out to the load before centrifugation and fraction 1 to 10 dyes SDS-PAGE points Analyse (fraction 1 is in bottom of the tube).Arrow shows the position of wheel virus antigen.Figure 45 B are to use rabbit polyclonal anti-rotavirus The Western blot analysis that antibody pair is carried out with identical fraction in (A).Figure 45 C be using the anti-VP2 antibody pair of rabbit polyclonal with (A) Western blot analysis that identical fraction is carried out in.

Figure 46 is shown by Iodixanol density gradient centrifugation to the class rotaviral particles comprising VP2, VP6 and VP7 Purifying.Figure 46 A are that the coomassie carried out to the load before centrifugation and fraction 1 to 10 dyes SDS-PAGE analysis (fractions 1 In bottom of the tube).Arrow shows the position of wheel virus antigen.Figure 46 B are to be resisted with (A) middle rank point identical using rabbit polyclonal The Western blot analysis of rotavirus antibody.Figure 46 C are to divide identical to use the anti-VP7 antibody of rabbit polyclonal with (A) middle rank Western blot analysis.

Figure 47 shows the class rotavirus comprising VP2, VP4, VP6 and VP7 by Iodixanol density gradient centrifugation The purifying of particle.For the coomassie of the load before centrifugation and fraction 1 to 10 dyeing SDS-PAGE analyses, (fraction 1 exists Figure 47 A Bottom of the tube).Arrow shows the position of wheel virus antigen.Figure 47 B are to use rabbit polyclonal anti-rotavirus antibody pair and (A) The Western blot analysis that middle identical fraction is carried out.Figure 47 C are using identical in the anti-VP7 antibody pair of rabbit polyclonal and (A) The Western blot analysis that fraction is carried out.

Figure 48 is shown by anti-VP4 specific ELISAs to the purified class colyliform comprising VP2, VP4, VP6 and VP7 The evaluation of VP4 contents in virion.

Figure 49 is shown comprising VP2 and VP6 (left part) and the warp for including VP2, VP4, VP6 and VP7 (right part) The cryo EM image of the class rotaviral particles of purifying.

Embodiment

It is the explanation on preferred embodiment below.

The present invention relates to include one or more rotavirus structural protein matter (i.e. class rotaviral particles, colyliforms Virus vlps or RLP) viruslike particle (VLP) and in plant produce class rotaviral particles (RLP) method.Therefore, Class rotaviral particles (RLP) can include one or more rotavirus structural protein matter.RLP can be bilayer or three Layer.

The present invention provide in part the method that class rotaviral particles (RLP) are produced in plant.Methods described can be with Part including one or more nucleic acid comprising following control regions to be introduced to plant or plant, the control region is in plant In nucleotide sequence that is active and being operatively connectable to encode one or more rotavirus structural protein matter. After this after the part of the plant or plant is cultivated under conditions of allowing the nucleic acid transient expression, RLP is thereby produced.

In addition, the present invention provide in part the side that class rotaviral particles (RLP) vaccine candidate object is produced in plant Method.Methods described can include：First nucleic acid, the second nucleic acid and the 3rd nucleic acid are introduced to the plant, the part of plant or plant In thing cell, first nucleic acid is included in active in the plant and is operatively connectable to encode the first rotavirus First control region of the first nucleotide sequence of structural protein matter, second nucleic acid be included in the plant in it is active simultaneously And it is operatively connectable to encode the second control region of the second nucleotide sequence of the second rotavirus structural protein matter, it is described 3rd nucleic acid is included in active in the plant and is operatively connectable to encode third round shape viral structural proteinses matter Trinucleotide sequence the 3rd control region.Allowing the bar of the first, second, and third nucleic acid transient expression after this The plant, the part of plant or plant cell are cultivated under part, thereby produces RLP.RLP can be individual layer, bilayer or three layers 's.

" rotavirus structural protein matter " can represent the rotavirus structural protein matter sequence from rotavirus separation What is arranged is all or part of, and it is present in any natural or anomaly rotavirus strain or separation strains.Therefore, term colyliform disease Malicious structural protein matter etc. is included during viral lifecycle by being mutated caused or response in selection pressure (example Such as, medicinal treatment, the expansion of host cell taxis or infectivity etc.) and caused Natural rotavirus structural protein matter sequence Variant.As understood by those skilled in the art, these rotavirus structural protein matter can also be produced using recombinant technique Sequence and its variant.

In addition, structural protein matter can include capsid protein (for example, VP2 and VP6) and/or surface protein (for example, VP4).The structural protein matter can also include such as VP7.

The non-limiting examples of rotavirus structural protein matter are rotavirus protein VP2, VP4, VP6 and VP7, And VP2, VP4, VP6 and VP7 fragment.Can be with VP2, VP4, VP6 and VP7 or VP2, VP4, VP6 used according to the invention Include with the non-limiting examples of the fragment of VP7 protein from rotavirus strain G9P [6], rotavirus A WA strains, colyliform disease Those VP2, VP4, VP6 of malicious A vaccines USA/Rotarix-A41CB052A/1988/G1P1A [8] strains and rotavirus SA11 strain With VP7 protein.

The example of VP2 structural protein matter is shown in SEQ ID NO：1 and SEQ ID NO：In 25 amino acid sequence, but This is not considered as restricted.In addition, VP2 structural proteins matter can include SEQ ID NO：1、SEQ ID NO：25 show The sequence gone out, or with them there is at least about 90-100% sequence similarities (to be included in any percentage phase in the range of these Like degree, such as there is 91,92,93,94,95,96,97,98,99% sequence similarity with them) sequence.In addition, VP2 structures Property protein can be by SEQ ID NO：13rd, it is nucleotide sequence coded shown in 14,25 or 45, or by having at least about with them 80-100% sequence similarities (be included in any percentage similarity in the range of these, such as with they have 81,82,83, 84th, 85,86,87,88,89,90,91,92,93,94,95,96,97,98,99% sequence similarity) sequence coding.

The example of VP4 structural protein matter is shown in SEQ ID NO：2 and SEQ ID NO：In 36 amino acid sequence, but This is not considered as restricted.In addition, VP4 structural proteins matter can include SEQ ID NO：2、SEQ ID NO：36 show The sequence gone out, or with them there is at least about 90-100% sequence similarities (to be included in any percentage phase in the range of these Like degree, such as there is 91,92,93,94,95,96,97,98,99% sequence similarity with them) sequence.In addition, VP4 structures Property protein can be by SEQ ID NO：15th, it is nucleotide sequence coded shown in 16,47,50 or 51, or by having extremely with them Few about 80-100% sequence similarities (be included in any percentage similarity in the range of these, such as with them with 81,82, 83rd, 84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99% sequence similarity) sequential coding.

The example of VP6 structural protein matter is shown in SEQ ID NO：3 and SEQ ID NO：In 31 amino acid sequence, but This is not considered as restricted.In addition, VP6 structural proteins matter can include SEQ ID NO：3、SEQ ID NO：31 show The sequence gone out, or with them there is at least about 90-100% sequence similarities (to be included in any percentage phase in the range of these Like degree, such as there is 91,92,93,94,95,96,97,98,99% sequence similarity with them) sequence.In addition, VP6 structures Property protein can be by SEQ ID NO：17th, it is nucleotide sequence coded shown in 18 or 46, or by there is at least about 80- with them 100% sequence similarity (be included in any percentage similarity in the range of these, such as with they have 81,82,83,84, 85th, 86,87,88,89,90,91,92,93,94,95,96,97,98,99% sequence similarity) sequential coding.

The example of VP7 structural protein matter is shown in SEQ ID NO：4 and SEQ ID NO：In 39 amino acid sequence, but This is not considered as restricted.In addition, VP7 structural proteins matter can include SEQ ID NO：4、SEQ ID NO：39 Hes SEQ ID NO：Sequence shown in 43, or with them there is at least about 90-100% sequence similarities (to be included in the range of these Any percentage similarity, such as with they have 91,92,93,94,95,96,97,98,99% sequence similarity) sequence Row.In addition, VP7 structural proteins matter can be by SEQ ID NO：19th, the nucleotide sequence shown in 20,48,49,52,53 or 54 Numbering, or by them there is at least about 80-100% sequence similarities (it is similar to be included in any percentage in the range of these Degree, such as there is 81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99% sequence with them Row similarity) sequential coding.

Can (it uses BLAST (basic Local Alignment search tools) 2.0 with TBLASTN programs by using BLASTP Algorithm) calculate amino acid sequence similarity or homogeneity.Technology for calculating amino acid sequence similarity or homogeneity is Well known to a person skilled in the art, also, the use of BLAST algorithm is described in ALTSCHUL et al. (1990, J Mol.Biol.215:403-410) and ALTSCHUL et al. (1997, Nucleic Acids Res.25:In 3389-3402).

Term " viruslike particle (VLP) " or " VLP " refer to following structures, and the structure self assembly forms and comprising one Or more structural protein matter (e.g., for example, rotavirus structural protein matter, such as, but not limited to VP2, VP4, VP6 and/ Or VP7 structural proteins matter).VLP comprising rotavirus structural protein matter may be additionally referred to as " rotavirus VLP ", " class wheel Shape virion (RVLP) ", " class rotaviral particles (RLP) ", " class rotaviral particles ", " RVLP " or " RLP ".VLP or RLP is generally morphologically similar to caused virion in infection with antigenicity, but lacks the hereditary information for being enough to replicate And thus it is non-infective.VLP can be produced in suitable host cell (including plant host cell).Thin from host Born of the same parents extract and separation and further after purification under the proper conditions, can be complete structure by VLP purifying.RLP can be individual layer, Bilayer or three layers of RLP.Can be by expressing rotavirus structural protein matter (such as a VP2 or VP6) acquisition individual layer RLP.It can be obtained by expressing two rotavirus structural protein matter (e.g., for example, by co-expressing both VP2 and VP6) Double-deck RLP, this can be carried out in the case where being with or without VP4.At least three rotavirus structural protein matter can be passed through While express (for example, VP2, VP6 and VP7 coexpression) and obtain three layers of RLP, this can be in the case where being with or without VP4 Carry out.VP4 coexpression causes to generate the particle with furcella, and it is similar with Natural rotavirus.Can by VP4 process or Cut to produce VP5 and VP8.This processing can use endogenous proteinase or by co-expressing suitable protease (for example, pancreas Protease, trypsin like proteases, serine protease, chymotrypsin-like proteinase, subtilopeptidase A) betide place In master.It is alternatively possible to during any step of RLP extraction flows or after RLP purifying, by adding suitable egg White enzyme is (for example, trypsase, trypsin like proteases, serine protease, chymotrypsin-like proteinase, hay bacillus egg White enzyme) processing VP4, to produce VP5 and VP8.

Every kind of rotavirus structural protein matter has different characteristic and size, and for being assembled into needed for RLP Amount is different.Term " rotavirus VLP ", " rotavirus viruslike particle (RVLP) ", " rotavirus viruslike particle (RLP) ", " rotavirus viruslike particle ", " RVLP " or " RLP " refers to include the structural egg of one or more rotavirus The viruslike particle (VLP) of white matter.The example of rotavirus structural protein matter can include but is not limited to VP2, VP4 (or VP5 and VP8), VP6 and VP7 structural protein matter.

Present invention also offers the method that RLP is produced in plant, wherein encoding the first colyliform viral structural proteinses matter The second rotavirus structural protein matter (example of first nucleic acid (the first nucleic acid) of (for example, VP2 or VP6 protein) and coding Such as, VP6 or VP2 protein) the second nucleic acid coexpression.In addition, coding third round shape viral structural proteinses matter is (for example, VP4 Or VP7) the 3rd nucleic acid can be co-expressed with the first and second nucleic acid, so as to co-expressed in plant first, second nucleic acid and 3rd nucleic acid.First nucleic acid, the second nucleic acid and the 3rd nucleic acid can be introduced into plant in same steps, or can be suitable Sequence is introduced in plant.By co-expressing the suitable protease of coding (for example, trypsase, trypsin like proteases, silk ammonia Pepsin, chymotrypsin-like proteinase, subtilopeptidase A) nucleic acid, VP4 can be processed or be cut in host Cut, to produce VP5 and VP8.It is alternatively possible to during any step of RLP extractions or after RLP purifying, by adding Enter satiable (satiable) protease (for example, trypsase, trypsin like proteases, serine protease, rotten albumen Enzyme sample protease, subtilopeptidase A) process VP4.

Furthermore, it is possible to the 4th core with coding fourth round shape viral structural proteinses matter (for example, VP7 or VP4 protein) Sour further translation table (encodes the second colyliform up to the first nucleic acid (the first colyliform viral structural proteinses matter of coding), the second nucleic acid Viral structural proteinses matter) plant with the 3rd nucleic acid (coding third round shape viral structural proteinses matter), so that first, Second nucleic acid, the third and fourth nucleic acid co-express in plant.By co-expressing the suitable protease of coding (for example, tryptose Enzyme, trypsin like proteases, serine protease, chymotrypsin-like proteinase, subtilopeptidase A) nucleic acid, can be with VP4 is processed or cut in host, to produce VP5 and VP8.It is alternatively possible to during any step of RLP extractions Or after RLP purifying, by adding satiable protease (for example, trypsase, trypsin like proteases, silk ammonia Pepsin, chymotrypsin-like proteinase, subtilopeptidase A) process VP4.

Furthermore, it is possible to expression is encoded into one or more rotavirus structural protein matter (for example, VP2 or VP6 albumen Matter) the first plant and the expression of the first nucleic acid to encode one or more rotavirus structural protein matter (such as but unlimited In VP6 or VP2 protein) the second nucleic acid the second plant hybridization, with produce coexpression be separately encoded VP2 and VP6 or respectively Encode the progeny plant (the 3rd plant) of VP6 and VP2 first and second nucleic acid.Furthermore, it is possible to by expression be separately encoded VP2 and VP6 is separately encoded VP6 and VP2 the 3rd plant and the expression of first and second nucleic acid and encodes one or more colyliforms diseases 4th plant hybridization of the 3rd nucleic acid of malicious structural protein matter (such as, but not limited to VP4 or VP7), to produce coexpression difference Encode the further progeny plant (the 5th plant) of VP2, VP6 and VP4 or VP7 first, second, and third nucleic acid.Can be with Using host protein enzyme, or by the suitable albumen of coexpression coding in one of plant of first, second, third or the 4th Enzyme is (for example, trypsase, trypsin like proteases, serine protease, chymotrypsin-like proteinase, bacillus subtilis protein Enzyme) nucleic acid, VP4 is processed or cut in plant, to produce VP5 and VP8.It is alternatively possible to appointing in RLP extractions During what step or after RLP purifying, by adding satiable protease (for example, trypsase, trypsin-like egg White enzyme, serine protease, chymotrypsin-like proteinase, subtilopeptidase A) process VP4.

As described in more detail below, one or more rotavirus structural protein matter can be encoded by expression The nucleic acid (the first nucleic acid) of (such as, but not limited to VP2, VP6 or VP7) produces RLP in plant.It can be co-expressed in plant Encode the second rotavirus structural protein matter (such as, but not limited to VP7, VP6 or VP2) the second nucleic acid.Furthermore, it is possible to 3rd nucleic acid of coexpression coding third round shape viral structural proteinses matter (such as, but not limited to VP6, VP7 or VP2) in plant. Nucleic acid, the second nucleic acid and the 3rd nucleic acid can be introduced into plant in same steps, or they can be sequentially introduced Into plant.Nucleic acid, the second nucleic acid and the 3rd nucleic acid can be introduced into plant with transient fashion or with stationary mode.

Furthermore, it is possible to the second core with the second rotavirus structural protein matter of coding (such as, but not limited to VP6 or VP7) Acid carrys out translation table up to the plant of the first nucleic acid of the first colyliform viral structural proteinses matter (for example, VP2 protein) of coding, so that So that both the first nucleic acid and second nucleic acid co-express in plant.Coding third round shape viral structural proteinses matter can be used 3rd nucleic acid of (such as, but not limited to VP7 or VP6) is further converted to the plant.

It is alternatively possible to reach VP6 or VP7 protein (the second core with the first nucleic acid of coding VP2 protein come translation table Acid) plant so that both the first nucleic acid and second nucleic acid co-express in plant.Coding third round shape virus can be used 3rd nucleic acid of structural protein matter (such as, but not limited to VP7 or VP6) is further converted to the plant.

Furthermore it is possible to turned with the 3rd nucleic acid of coding third round shape viral structural proteinses matter (for example, VP4 or VP7) Change the first and second nucleic acid that expression encodes the first and second rotavirus structural protein matter (for example, VP2 and VP6 protein) Plant.By co-expressing the suitable protease of coding (for example, trypsase, trypsin like proteases, serine stretch protein Enzyme, chymotrypsin-like proteinase, subtilopeptidase A) nucleic acid, VP4 can be processed or be cut, with produce VP5 and VP8.It is alternatively possible to during any step of RLP extractions or after RLP purifying, by adding satiable albumen Enzyme is (for example, trypsase, trypsin like proteases, serine protease, chymotrypsin-like proteinase, bacillus subtilis protein Enzyme) process VP4.

Present invention also offers the method that RLP is produced in plant, methods described includes drawing one or more nucleic acid Enter into plant, the part of plant or plant cell, one or more nucleic acid encodes one or more rotavirus Structural protein matter and it is operatively connectable to active control region in plant and one or more compartments Target sequence and/or amplification element.Then, cultivate plant under conditions of one or more expression of nucleic acid is allowed, plant The part of thing or plant cell, thus produce RLP.One or more rotavirus structural protein matter can be VP2, VP4 (or VP5 and VP8), VP6, VP7, VP2, VP4 (or VP5 and VP8), VP6, VP7 fragment or its combination.

Present invention also offers the RLP for including one or more rotavirus structural protein matter, the rotavirus Structural protein matter is such as, but not limited to VP2, VP4 (or VP5 and VP8), VP6, VP7 or its combination.Can be by such as of the invention One or more methods provided produce RLP.

Any suitable method can be used, such as density gradient centrifugation or size-exclusion chromatography detect RLP appearance. RLP structure and size can be assessed for example, by electron microscope or by size-exclusion chromatography.

For size-exclusion chromatography, can by Extraction buffer by it is chilled-extruding vegetable material sample Product homogenization (Polytron), to extract total soluble protein from plant tissue, and insoluble material is removed by centrifugation.With It is also likely to be beneficial that ice-cold acetone or PEG, which carry out precipitation,.Soluble protein is quantified, and extract is passed through Sephacryl^TMPost, such as Sephacryl^TMS500 posts.Blue Dextran 2000 can be used as calibration standard items. After chromatography, fraction can further be analyzed by Western blot, to determine the protein complement (protein of fraction complement)。

For example, the fraction of separation can be supernatant (if by centrifuging, depositing or precipitate) or filtrate (if passed through Filter), and protein or superstructure protein are enriched, such as such as nanotube, nanosphere, or higher-order, higher molecular weight Particle, such as individual layer (sl), the RLP of double-deck (dl) or three layers (tl).

Can be for example, by extra centrifugation step, precipitation, chromatographic step (for example, size-exclusion chromatography, ion exchange color Spectrum, affinity chromatography), tangential flow filtration or combinations thereof, separated fraction is processed further, with to protein, The particle of superstructure protein or higher-order is separated, purified, concentrated or combinations thereof.Can for example, by natural or SDS-PAGE, using the immunoassay of appropriate detection antibody, Capillary Electrophoresis, electron microscope or those skilled in the art are shown And any other method being clear to confirms the presence of protein, superstructure protein or the higher-order particle (such as RLP) purified.

The RLP according to caused by the present invention can be purified or partial purification is from plant, the part of plant or vegetable material, Huo Zheqi Method as known to those skilled can be used to be applied as Oral vaccine.

RLP purifying can include gradient centrifugation, for example, sucrose, Iodixanol, OptiPrep^TMOr cesium chloride (CsCl) is close Degree gradient can be used for purifying or partial purification RLP from the biomass of inverted plant.As shown in such as Figure 45, it can make RLP and/or expression rotavirus structural protein matter are purified with Iodixanol step gradient or Iodixanol continuous gradient.

It is critically important to show that calcium (Ca2+) concentration converts to three layers of particle (TLP) to double layer particle (DLP), and it is poison Strain dependence (see, e.g. Martin et al. .Journal of Virology, Jan 2002, the document is by quoting simultaneously Enter herein).Outer capsid proteins matter is from TLP complete loss (the TLP effects of raising one's hat) in [Ca²⁺] nanomole scope in occur.Cause This, RLP purifying and/or extraction can be carried out in the presence of calcium, and gradient centrifugation step can have calcium In the case of carry out, for example, CaCl be present₂In the case of carry out.For example, CaCl2 concentration can 1mM to 1000mM it Between, or can be any amount between them, such as 2,3,4,5,6,7,8,9,10,11,12,13,14,15,20,25,30, 40、50、100、150、200、250、300、350、400、450、500、50、600、650、700、750、800、850、900、 950mM or any amount between them.

The processing of minimum degree can be carried out to plant or phytoclasts.Term " processing of minimum degree " refers to following plants Material, for example, by partial purification to obtain plant extracts, homogenate, plant homogenates part etc. (i.e. minimally process) , include plant of protein and/or RLP of interest or part thereof.It is thin that partial purification may include but be not limited to destruction plant Born of the same parents' structure, thus produce comprising soluble plant ingredient and insoluble plant composition (can for example, by but be not limited to from The heart, filtering or its combination separation) composition.In this respect, vacuum or centrifugation extraction can be used and be easily achieved in leaf The endocrine protein in the space between cells of piece or other tissues, or tissue can be extracted under stress, and this is by roller or grinds Mill etc. is carried out, to be extruded out of space between cells or discharge protein.The processing of minimum degree can also include to soluble protein The preparation of the crude extract of matter, because these preparations may be by with the insignificant pollution from secondary plant products.In addition, most The processing of small degree can include carrying out extraction with aqueous solution to soluble protein from blade, then be carried out with any suitable salt Precipitation.Other methods can include extensive dipping and sap extraction, to enable extract directly to use.It can use and appoint What suitable method (for example, mechanical extraction or biochemical extraction) purifies or extracted RLP.

One or more rotavirus structural protein matter can be with up to 2g for relative per kilogram plant fresh weight Amount is synthesized.For example, the amount of the structural protein matter of synthesis can for relative per kilogram plant fresh weight 1 between 2g, or Person is any amount between them, as be 1.0 for relative per kilogram fresh weight, 1.1,1.2,1.3,1.4,1.5,1.6,1.7, 1.8th, 1.9,2g or any amount between them.For example, for structural protein matter can be with relative per kilogram plant fresh weight Up to 1.54g amount synthesis.

In addition, RLP can be synthesized with being up to 1.5g amount for relative per kilogram plant fresh weight.For example, the RLP of synthesis Amount can be for relative per kilogram plant fresh weight 0.5 to any amount between 1.5g, or between them, it is such as relatively every Be 0.5 for kilogram plant fresh weight, 0.6,0.7,0.8,0.9,1.0,1.1,1.2,1.3,1.4,1.5g.For example, RLP can be with It is synthesized with respect to the amount that 1.1g is up to for per kilogram plant fresh weight.

It can measure RLP's defined above for example, by dynamic light scattering (DLS) or electron microscope (EM) technology Size (i.e. diameter), the size or are any size between them between 110nm generally 50.For example, complete RLP The size of structure can be in the range of about 70nm to about 110nm, or any size between them, for example, 75nm, 80nm, 85nm, 90nm, 95nm, 100nm, 105nm or any size between them.

It is described nucleotide sequence coded one or more present invention also offers the nucleic acid comprising following nucleotide sequence Rotavirus structural protein matter is simultaneously operatively connectable to active control region in plant.It is close such as people can be directed to The use of numeral or the use of vegetable codon optimize to nucleotide sequence.In addition, one or more rotavirus knots Structure protein can be operatively connectable to one or more amplification element.In addition, one or more colyliform diseases Malicious structural protein matter can be operatively connectable to one or more compartment targeting sequence.Pass through the nucleotides sequence One or more rotavirus structural protein matter of row coding can be such as VP2, VP4, VP6 or VP7.In addition, pass through Nucleotide sequence coded one or more rotavirus structural protein matter can come from for example, organizing A to times for organizing G What rotavirus, but the rotavirus more preferably from group A.In addition, by described nucleotide sequence coded one or more Multiple rotavirus structural protein matter can come from having from G1 to G27 and from P1 to P34 (more preferably from G1 to G19 and From P1 to P27) G- types and P-type any combination of genotype any rotavirus strain, including but not limited to G1P [8], G2P [4], G2P [8], G3P [8], G4P [8], G9P [6], G9P [8], rotavirus A WA strains, rotavirus A vaccines USA/ Rotarix-A41CB052A/1988/G1P1A [8] strains or rotavirus SA11 strain.

For nucleotide sequence mentioned in the present invention, if the nucleotide sequence and one or one or more such as this paper institutes The nucleotide sequence of definition or the complementary series of nucleotide sequence hybridize under stringent hybridization condition, then the nucleotide sequence can be with It is and the complementary series of the sequence or the sequence " substantially homologous ", " substantially similar " or " substantially same ".To multiple For several sequences, when any amount (example at least about between 70% or 70% to 100% or between them in nucleotides Such as, 70,72,74,76,78,80,82,84,86,88,90,92,94,96,98,100% or any amount between them) in core When being matched on the given length of nucleotide sequence, the sequence is " substantially homologous ", " substantially similar " or " substantially same ", But on condition that these homologous sequences show one or more kinds of property of the coded product sequence or as described herein Matter.

For example, the invention provides separated polynucleotides, it, which is included, encodes one or more rotavirus structures Property protein nucleic acid, the rotavirus structural protein matter and such as SEQ ID NO：13、14、15、16、17、18、19、 20th, the sequence defined in 45,46,47,49,50,51,52,53,54 have at least 60%, 65%, 70%, 75%, 80%, 85%th, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% or the homogeneity of any amount between them.The polynucleotides can be by any side as known in the art Method has carried out the polynucleotides of people's codon optimization.

In addition, the invention provides the RLP for including rotavirus structural protein matter, the rotavirus structural protein Matter is for example encoded by following nucleic acid, the nucleic acid and such as SEQ ID NO：13、14、15、16、17、18、19、20、 45th, the sequence defined in 46,47,49,50,51,52,53,54 have at least 60%, 65%, 70%, 75%, 80%, 85%th, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% or the homogeneity of any amount between them.

The sequence similarity or homogeneity can be determined using nucleotide sequence comparison program, as carried in DNASIS For (wherein use such as, but not limited to following parameters：Gap Penalty (GAP penalty) is 5, # tops diagonal is 5, solid It is that 10, k first (k tuple) is that 2, floating breach (floating gap) is 10 to determine Gap Penalty, and 5) window size is.But This area it is also known that other sequence alignment methods for comparing, such as Smith＆Waterman algorithm (1981, Adv.Appl.Math.2:482), Needleman＆Wunsch algorithm (J.Mol.Biol.48:443,1970)、Pearson& Lipman algorithm (1988, Proc.Nat'l.Acad.Sci.USA 85:2444) and these algorithms computerization implement (GAP, BESTFIT, FASTA and BLAST, can be obtained by NIH), or manually compare and visually inspect (see, e.g., Current Protocols in Molecular Biology, Ausubel et al. edit .1995supplement), or use Southern or Northern hybrid methods under stringent condition are (referring to Maniatis et al. is in Molecular Cloning (A Laboratory Manual), Cold Spring Harbor Laboratory, described in 1982).Preferably, substantially together The sequence in source shows at least about 80% on the given length of molecule, and most preferably at least about 90% sequence is similar Degree.

A kind of example of such stringent hybridization condition can be that (about 16-20 is small for hybridized overnight in 4 × SSC at 65 DEG C When), then at 65 DEG C with 0.1 × SSC clean 1 hour, or at 65 DEG C with 0.1 × SSC cleaning twice, every time 20 or 30 minutes.Alternatively, a kind of Exemplary stringent hybridization condition can be hybridized at 42 DEG C in 50% formamide, 4 × SSC At night (16-20 hours), then cleaned 1 hour with 0.1 × SSC at 65 DEG C, or cleaned twice with 0.1 × SSC at 65 DEG C, 20 or 30 minutes every time；Or can be in Church phosphate aqueous buffer solutions (7%SDS at 65 DEG C；0.5M NaPO4 delay Fliud flushing, pH 7.2；10mM EDTA) in hybridized overnight (16-20 hours), and at 50 DEG C with 0.1 × SSC, 0.1%SDS clean Twice, 20 or 30 minutes every time, or cleaned twice with 2 × SSC, 0.1%SDS at 65 DEG C, 20 or 30 minutes every time (for only Special sequence area).

The nucleic acid for encoding colyliform viral structural polypeptide can be described as " rotavirus nucleic acid ", " rotavirus nucleosides Acid sequence ", " rotavirus nucleic acid " or " rotavirus nucleotide sequence ".For example, it can will include one or more colyliforms The viruslike particle of viral structural proteinses matter or the structural polypeptide of rotavirus be described as " rotavirus VLP ", " RVLP " or " RLP ", but this is not construed as limiting.

A variety of biologies show the preference inserted using specific codon encoding particular amino acid in the peptide chain of extension. Codon is preferably or codon preference (difference that codon uses between biology) is undertaken by the degeneracy of genetic code, and by It is recorded in well in a variety of biologies.Codon preference is generally related to the translation efficiency of mRNA (mRNA), itself so that recognized For the property depending on the codon to be translated and the availability of specific transfer RNA (tRNA) molecule etc..It is selected in cell The tRNA selected advantage is typically the reflection to codon most-often used in peptide symthesis.Therefore, codon optimization can be based on Gene is adjusted, to realize the optimal gene expression in given biology.The protein of Optimized Coding Based heterogenous expression The process of nucleotide sequence is probably to improve the important step of expression yield.The requirement of optimization can include improving outside host's production The step of carrying out the ability of protein.

" codon optimization " is defined as：May more often it be used or the most frequently used by using in the gene of another biology or species Codon replace native sequences it is at least one, carry out modification of nucleic acids sequence more than one or significant amount of codon, to carry The high expression in cell of interest.A variety of species show the certain preference to some codons of specific amino acids.

The present invention includes the synthetic polyribonucleotides sequence for having already passed through codon optimization, for example, being directed to people's codon Using or vegetable codon use the sequence that optimized.It is then possible to the polynucleotides through codon optimization are expressed in plant Sequence.More specifically, the sequence optimized for the use of people's codon or the use of vegetable codon can be expressed in plant Row.In the case where being not wishing to be bound by theory, it is believed that for the sequence that people's codon optimized improve the guanine of sequence- Cytosine content (G/C content), and improve the expression yield in plant.

Different codon-optimization techniques as is generally known in the art be present, poorly efficient protein coding region is translated for improving Translate dynamics.These technologies depend on identification and used for the codon of specific host biology.If in the biology Specific gene or sequence should be expressed, the coded sequence of those this genoids and sequence will be modified, so as to use in host organism more Conventional codon replaces the codon of sequence of interest.

The structural egg of rotavirus can be expressed in comprising DNA or the expression system of rna expression system based on virus White matter or polypeptide, the expression system are such as, but not limited to the expression cassette based on cowpea mosaic virus (comovirus) and are based on The amplification element of geminivirus infection (geminivirus).

Expression system as described herein can be included based on two partitivirus (bipartite virus) or with two points of bases Because of group (bipartite genome) viral expression cassette.For example, two partitivirus can be Comoviridae (Comoviridae family's).The category of Comoviridae includes Comovirus (Comovirus), compacted biography disease Poison category (Nepovirus), pulse family Tobamovirus (Fabavirus), cherry rasp leaf virus category (Cheravirus) and satsuma orange are short Contracting Tobamovirus (Sadwavirus).Comovirus includes cowpea mosaic virus (CPMV), the serious mosaic virus of cowpea (CPSMV), pumpkin mosaic virus (SqMV), red clover mottle virus (RCMV), bean pod mottle virus (BPMV), turnip ring Pinta poison (TuRSV), echtes ackerbohnemosaik virus (BBtMV), broad bean dyeing viral (BBSV), radish mosaic virus (RaMV).Comprising Can be used for the example of the cowpea mosaic virus RNA-2 sequences of the enhancer element of many aspects of the present invention includes but unlimited In：CPMV RNA-2 (GenBank accession number No.NC_003550), RCMV RNA-2 (GenBank accession number No.NC_ 003738), BPMV RNA-2 (GenBank accession number No.NC_003495), CPSMV RNA-2 (GenBank accession number No.NC_ 003544), SqMV RNA-2 (GenBank accession number No.NC_003800), TuRSV RNA-2 (GenBank accession number No.NC_013219.1), BBtMV RNA-2 (GenBank accession number No.GU810904), BBSV RNA2 (GenBank accession number No.FJ028650), RaMV (GenBank accession number No.NC_003800).

Piece the severed finger RNA-1 and RNA-2 of two points of cowpea mosaic virus rna gene groups.RNA-1 codings participate in the albumen replicated Matter, and protein and two capsid proteins needed for RNA-2 Codocytes-cell movement.Any suitable base can be used In the box of cowpea mosaic virus, including CPMV, CPSMV, SqMV, RCMV or BPMV, for example, the expression cassette can be based on CPMV。

" expression cassette " refers to following nucleotide sequence, and the nucleotide sequence includes nucleic acid of interest, and the nucleic acid is in For being transcribed in host cell under the suitable promoter of the nucleic acid of interest or the control of other controlling elements, and Operationally (operatively) it is connected to the suitable promoter or other controlling elements.

Expression system can also include the amplification element from geminivirus infection (geminivirus), for example, yellow from beans The amplification element of dwarf virus (BeYDV).BeYDV belongs to Mastreviruses category (it is adapted to dicotyledon).BeYDV is Single partitivirus (monopartite) with single stranded circle DNA genomes, and it can be copied to very by rolling ring mechanism High copy number.The amplicon dna carrier system in BeYDV sources has been used for carrying out the quick, albumen of high yield in plant Matter produces.

As it is used herein, term " amplification element " refers to one or more long genes comprising geminivirus infection genome Between between area (long intergenic region) or long gene repeat (long intergenic repeat, LIR) at least The nucleic acid fragment of a part.As it is used herein, " long intergenic region " or " being repeated between long gene " refers to comprising rep binding sites Long intergenic region region, the rep binding sites can mediate the excision that is carried out by geminivirus infection Rep protein and multiple System.In some respects, the nucleic acid fragment comprising one or more LIR can also be included between the short gene of geminivirus infection genome Area (SIR) between area or mini gene.As it is used herein, " short intergenic region " or " area between mini gene " refers to complementary strand (Mastrevirus short IR (SIR)).Can be with any suitable amplification element from geminivirus infection used herein. See, e.g., WO2000/20557；WO2010/025285；Zhang X. et al. (2005, Biotechnology and Bioengineering, Vol.93,271-279), Huang Z. et al. (2009, Biotechnology and Bioengineering, Vol.103,706-714), Huang Z. et al. (2009, Biotechnology and Bioengineering,Vol.106,9-17)；Document above is incorporated herein by reference).If used in construct more In a LIR, for example, two LIR, then promoter, CMPV-HT areas and nucleotide sequence of interest and terminator are by two Each surrounding in LIR.In addition, amplification element can be for example from Halley-Stott et al. (2007) Archives of Virology152:Sequence disclosed in 1237-1240, with Gen Bank accession number DQ458791, (it is incorporated by reference into this for it Text) registration.Nucleic acid fragment comprising LIR is the nucleotides 2401~2566 and 1~128 of connection.Nucleic acid fragment comprising SIR is Nucleotides 1154~1212.

As described herein, entered by the agroinfiltration of tobacco Ben Saimushi (Nicotiana benthamiana) blade The capable common delivering that carrier is supplied to the carrier from beans yellow dwarf virus (BeYDV) and Rep/RepA result in efficient multiple System expands and powerful protein production.

Expression cassette based on cowpea mosaic virus and it can be contained in different loads from the amplification element of geminivirus infection On body, or, each part can be contained in a carrier.If, can be by first and using two carriers Two carriers are introduced in plant cell at the same time or separately.

Rdrp virus can also be included in expression system as described herein, to improve the table of nucleic acid of interest Reach.The non-limiting examples of replicase are to encode BeYDV Rep and RepA BeYDV replicase (pREP110) (C2/C1； Huang et al., 2009, Biotechnol.Bioeng.103,706-714；The document is incorporated herein by reference). Halley-Stott et al. (2007) Archives of Virology 152:The another of replicase is disclosed in 1237-1240 Individual non-limiting examples, it is registered with Gen Bank accession number DQ458791 (it is incorporated herein by reference).Include C1:C2 bases The nucleic acid fragment of cause is nucleotides 1310 to 2400.

" coexpression ", which refers to two or more nucleotides sequence and be listed in the roughly the same time, to be expressed in plant and should In the identical tissue of plant.However, nucleotide sequence need not be in identical temporal expressions.On the contrary, two or more cores Nucleotide sequence is to make coded product have an opportunity to express in a manner of interaction.Using transient expression system can co-express two or Nucleotide sequence more than two, wherein under conditions of two sequences are expressed the roughly the same time by two kinds or more A variety of sequences are incorporated into plant.It is alternatively possible to use the coding protein of interest that platform plant is introduced with transient fashion Other sequences of (for example, one or more rotavirus structural protein matter), in a stable manner conversion include nucleotides The platform plant of one of sequence.

The correct folding of protein is probably weight to the stability of protein, the formation of polymer, RLP formation and function Want.The folding of protein can be influenceed by one or more factors, and these factors include but is not limited to：The sequence of protein Row, the relative abundance of protein, the intracellular degree of crowding, can with fold, partially folded or unfolded protein be combined or To the availability of their instantaneous related co-factors.In addition, the endophytic compartment or subcompartment of marking protein can influence egg The folding of white matter and expression.

Can in genetically modified plants by agroinfiltration by one or more rotavirus structural protein matter Express targeted to specific palnt cell compartments and/or subcompartment.Compartment or subcompartment can be, for example, plastid, endoplasmic reticulum (ER), chloroplaset or apoplast.In the case where being not wishing to be bound by theory, compartment or subcompartment targeting can by protein to Accumulation in the compartment or subcompartment that are targetted is improved to more than cytoplasmatic accumulation.Compartment or subcompartment accumulation can be with protected proteins Matter is from the proteasome degradation present in cytoplasm and/or allows it to build up to higher concentration without influenceing plant cell Function.

Therefore, expression cassette or carrier can be changed, to be adapted for carrier or the colyliform disease from vector expression Malicious structural protein matter or Peptide T are directed at target compartment or subcompartment in plant.

For example, can by express rotavirus structural protein matter or polypeptide include can be with the class capsule of plastid The part (especially, the transporting mechanism of thylakoid membrane) of body membrane interaction, is changed to expression cassette or carrier, so that it is suitable Together in targeting plastid.This interaction can cause rotavirus structural protein matter or polypeptide defeated from the cytoplasm for expressing it Enter to plastid.In the case where being not wishing to be bound by theory, the correct folding from cytoplasmic input mechanism for protein comes Say to be probably important.It should be appreciated that can be changed to expression cassette or carrier, with make it suitable for targetting plastid in itself from And turn into inverted state, and the expression of rotavirus structural protein matter or polypeptide can occur in plastid completely.

Term " targeting sequence ", which refers to targeting sequence, can be included in carrier or expression cassette.Such targeting sequence can be turned over Be translated into the peptide of the target compartment or subcompartment (for example, plastid) that carrier or its product are guided into plant.For example, it is used for egg White matter is well known in the art targeted to the plastid signal peptide (being also known as in the art " plastid transit peptides ") of plastid.Can Non-limiting examples with the plastid transit peptides used are rbcs1-cTP.The suitable example of chloroplast transit peptide sequence is next From the Rubisco little subunits gene (rbcS1) of such as potato (Solanum tuberosum).

Therefore, rotavirus structural protein matter or polypeptide can include identical with the remainder of polypeptide or protein next Source or heterologous signal peptide.Term " signal peptide " is well known in the present art, and typically refers to short (about 5-30 amino Acid) amino acid sequence, it is generally found in the N-terminal of polypeptide, and the polypeptide that can instruct newly to translate is indexed into specific cells device, The specific domain of polypeptide chain is assisted relative to the positioning in other structures domain.As non-limiting examples, the signal peptide can be with By the indexing of protein targeted to endoplasmic reticulum, and/or the N-terminal proximal structure domain of nascent polypeptide is assisted relative to film anchoring domain Positioning, so as to aid in the cutting of mature protein (for example, rotavirus structural protein matter, but this is not construed as limiting) And folding.

Signal peptide (SP) can be it is natural for protein or virus protein, or signal peptide can be relative to It is heterologous for the protein or the primary sequence of virus protein to be expressed.For example, the day of rotavirus structural protein matter Right signal peptide can be used for expressing rotavirus structural protein matter in botanical system.

Signal peptide can also be non-natural, for example, from protein, virus protein or except rotavirus protein it Outer viral natural structure protein, or the polypeptide from plant, animal or bacterium.The signal peptide that can be used it is non- Limitative examples are clover (alfalfa) protein disulfide isomerase (PDI SP) (accession number No.Z11499 nucleotides 32-103).In addition, signal peptide can be lacked or be truncated completely.Truncate or its various part of speech form represents to lack from signal peptide 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%th, 80%, 85%, 90%, 95%, 100% or any amount between them amino acid residue.Preferably, the ammonia of truncation Base acid residue is continuous, and truncates and occur forward from second methionine.

Present invention thus provides the structural egg of rotavirus for including natural, non-native signal peptide or the signal peptide of truncation White matter, e.g., such as VP2, VP4, VP6 and/or VP7, and provide the nucleic acid for encoding these rotavirus structural protein matter.

Can be in any suitable plant host converted through nucleotide sequence of the present invention, construct or carrier Express the genetic constructs of one or more than one of the present invention.The example of suitable host includes but is not limited to crops, bag Include clover, rapeseed, Btassica (Brassica spp.), corn, Nicotiana (Nicotiana spp.), potato, ginseng, Pea, oat, rice, soybean, wheat, barley, sunflower, cotton etc..

1,2,3,4 or 5 binary plasmid carrier can be used to encode the nucleotides sequence of colyliform viral structural proteinses matter Column jump is entered in plant host.Therefore, each binary plasmid carrier can include 1,2,3,4 or 5 coding colyliform virus structure The nucleotide sequence of property protein.

One or more genetic constructs of the present invention can also include 3' non-translational regions.3 ' non-translational regions, which refer to, to be included down The Gene Partial of DNA segment is stated, the DNA segment contains polyadenylation signal and can have mRNA processing or gene expression Imitate any other Regulate signal carried out.The feature of polyadenylation signal, which is generally placed at, to add to 3 ' ends of mRNA precursor Polyadenylic acid chain is effectively carried out.Polyadenylation signal generally passes through the homology with the AATAAA-3 ' of canonical form 5 ' In the presence of identifying, but make a variation and quite a few see.The non-limiting examples in 3rd ' suitable area are the polyadenylic acids containing following gene Change the 3 ' non-translational regions through transcription of signal, the gene is：Agrobacterium (Agrobacterium) tumorigenesis (Ti) plasmid gene (such as nopaline synthase (NOS) gene), plant gene (such as soybean storage protein gene), ribulose-1,5-bisphosphate, 5- diphosphonic acid carboxylations Gene (the ssRUBISCO of enzyme little subunit；US 4962028, it is incorporated herein by reference), in US 7125978, (it passes through Be incorporated herein by reference) described in be used for regulate and control plastocyanin expression promoter.

When needing, one or more genetic constructs of the invention can also include other enhancer (translational enhancers Or transcriptional enhancer).Enhancer can be located at the 5 ' or 3 ' of the sequence that will be transcribed.Enhancer region is those skilled in the art Known, it can include ATG initiation codon, neighbouring sequence (adjacent suquences) etc..Initiation codon is present When, it may be at the state for the reading frame for meeting coded sequence (" inframe "), be turned over providing the correct of the sequence through transcription Translate.

Ti-plasmids, Ri plasmids, plant viral vector, directly delivered DNA, microinjection, electroporation etc. can be used to incite somebody to action this The construct of invention is incorporated into plant cell.On the summary of these technologies, see, for example, Weissbach and Weissbach,Methods for Plant Molecular Biology,Academy Press,New York VIII,pp 421-463(1988)；Geierson and Corey, Plant Molecular Biology, second edition (1988)；And Miki And Iyer, Fundamentals of Gene Transfer in Plants, In Plant Metabolism, second edition, DT.Dennis,DH Turpin,DD Lefebrve,DB Layzell(eds),Addison Wesly,Langmans Ltd.London,pp.561-579(1997).Other methods include direct DNA intakes, the use of liposome, electroporation (for example, Use protoplast), microinjection, micropellet bombardment (microprojectile) or whisker (whiskers) and vacuum penetrate into (vacuuminfiltration).See, e.g., Bilang et al. (Gene 100:247-250 (1991)), Scheid et al. (Mol.Gen.Genet.228:104-112,1991), Guerche et al. (Plant Science 52:111-116,1987)、 Neuhause et al. (Theor.Appl Genet.75:30-36,1987), Klein et al. (Nature 327:70-73 (1987))；Howell et al. (Science 208:1265,1980), Horsch et al. (Science 227:1229-1231, 1985), DeBlock et al. (Plant Physiology 91:694-701,1989)、Methods for Plant Molecular Biology (Weissbach and Weissbach are edited, Academic Press Inc., 1988), (Schuler and Zielinski are edited Methods in Plant Molecular Biology, Academic Press Inc.,1989)、Liu and Lomonossoff(J Virol Meth,105:343-348,2002), United States Patent (USP) No.4945050；5036006；With 5100792, the U.S. Patent Application Serial Number No.08/ that May 10 nineteen ninety-five submits (all of above document is by drawing by the U.S. Patent Application Serial Number No.07/951715 that 438666 and 1992 on Septembers are submitted for 25 With being incorporated herein).

Transient expression

In the case where being not wishing to be bound by theory, the protein concentration and ratio of different rotavirus structural protein matter Example is probably important for RLP packaging efficiency.Therefore, the multiplicity of infection and time are for the egg in control plant It is probably important for white matter concentration and RLP overall package efficiency.

The construct of the present invention can be in transient expression in the part of plant or plant.Dependent on recombinational agrobacterium Outside the chromosome of (Agrobacterium tumefaciens) in plant, the part of plant or plant cell (epichromosomal) transient expression system of expression, which can be used for expressing, is targeted to multiple cellular compartments or subcompartment Rotavirus structural protein matter.Transient expression system allows for high speed of production.In addition, in recombinational agrobacterium in plant In a couple of days after middle infiltration, can obtain a large amount of protein (Rybicki, 2010；Fischer et al., 1999).Length can also be expressed Gene order, and more than one gene is expressed simultaneously in same cell, so as to allow efficient group of polymer protein Fill (Lombardi et al., 2009).

The nucleotide sequence of coding colyliform viral structural proteinses matter can be transferred in plant host, to 1,2,3,4 or 5 Agrobacterium (Agrobacterium tumefaciens) bacterial strain of individual conversion.

However, during transient expression, PTGS may limit the expression of heterologous protein in plant.It is heavy The common table of silent repressor (such as, but not limited to the Nss from tomato spotted wilf virus (Tomato spotted wilt virus)) Up to the selective degradation (Brigneti et al., 1998) that can be used for offsetting transgenosis mRNA.Alternative silencing suppressor is at this It is known in field, and can be used as described herein (Chiba et al., 2006, Virology 346:7- 14；The document is incorporated herein by reference), such as, but not limited to HcPro, TEV-p1/HC-Pro (tobacco etch virus-p1/ HC-Pro), BYV-p21, the p19 (TBSV p19) of tomato bushy stunt virus, tomato crinkle virus capsid protein matter (TCV-CP), The 2b (CMV-2b) of cucumber mosaic virus, Potyvirus X p25 (PVX-p25), Potyvirus M p11 (PVM-p11), Potyvirus S p11 (PVS-p11), blueberry wilt virus pl6 (BScV-pl6), the p23 (CTV- of Citrus tristeza virus P23), the p24 (GLRaV-2p24) of grapevine leaf roll joint virus -2, grapevine virus A p10 (GVA-p10), grapevine disease Malicious B P14 (GVB-pl4), the p10 (HLV-p10) of root of Dahurain angelica latent virus or the common latent virus of garlic P16 (GCLV- p16).Therefore, silencing suppressor (for example, HcPro, TEV-p1/HC-Pro, BYV-p21, TBSV p19, TCV-CP, CMV-2b, PVX-p25、PVM-p11、PVS-p11、BScV-p16、CTV-p23、GLRaV-2p24、GBV-p14、HLV-p10、 GCLV-p16 Or GVA-p10) can be with one or more rotavirus structural protein matter (for example, VP2, VP4, VP6 or their group Close) co-express together, to further ensure that, high-caliber protein produces in the part of plant or plant.

Present invention also offers method as described above, wherein, other (second, third, the 4th or the 5th) nucleotides Sequence is expressed in plant, encodes others (second, third, the 4th or the 5th) nucleotide sequence operability of silencing suppressor Ground and others active in plant (second, third, the 4th or the 5th) control region is connected.Encode silencing suppressor Nucleotide sequence can be such as Nss, HcPro, TEV-p1/HC-Pro, BYV-p21, TBSVp19, TCV-CP, CMV-2b, PVX-p25、PVM-p11、PVS-p11、BScV-p16、CTV-p23、GLRaV-2p24、GBV-p14、HLV-p10、GCLV-p16 Or GVA-p10.

As described below, construct of the present invention can be expressed using instant expression method (referring to Liu and Lomonossoff,2002,Journal of Virological Methods,105:343-348；The document is by quoting simultaneously Enter herein).It is alternatively possible to using the instant expression method based on vacuum, such as Kapila et al., 1997, (document is by drawing With being incorporated herein) it is described.These methods can include (such as, but not limited to) Agrobacterium inoculation (Agro-inoculation) or Agroinfiltration (Agro-infiltration), the method for syringe infiltration (syringe infiltration), it is also possible to Other transient approach used as described above.Infiltrated by Agrobacterium inoculation, agroinfiltration or syringe, comprising desired The Agrobacterium mixture of nucleic acid enters histocyte gap, for example, leaf, plant gas first portion (including stem, leaf and flower), plant Other parts (stem, root, flower) or whole plant.After permeate through epidermis, Agrobacterium infection t-DNA is copied and transferred them to thin Born of the same parents.T-DNA is transcribed in a manner of free, and mRNA is translated, so as to cause that albumen of interest is produced in infected cell Matter, but it is instantaneous that t-DNA, which is pierced into core,.

To assist to differentiate inverted plant cell, the construct of the present invention can further be operated, with bag Include into plant and mark may be selected.Useful optional mark includes providing the enzyme to the resistance of chemical substance, the chemistry Material such as antibiotic, for example, gentamicin, hygromycin, kanamycins；Or herbicide, such as cremart (phosphinothrycin), glyphosate, chlorine sulphur are grand etc..Similarly, it can use that can produce can be by color change (such as GUS (beta-glucuronidase)) or photism (such as luciferase element and GFP) come the enzyme of compound that differentiates.

Genetically modified plants, plant cell or the seed of genetic constructs containing the present invention are also considered as the one of the present invention Part.It is also known in the art from the method for Plant cell regeneration whole plant.Usually, in suitable culture medium Inverted plant cell is cultivated, the culture medium can include selective reagent (such as antibiotic), wherein mark may be selected For aiding in differentiating inverted plant cell.Once form callus, then can be according to known method by using suitable Plant hormone promote bud to be formed, and the bud is transferred in root media with aftergrowth.Then, the plant can quilt Repeated from generation to generation for establishing, this can be realized from seed or be realized using vegetative reproduction technology.Genetically modified plants can also be without using Tissue cultures and produce.

In this application, the use of term " control region ", " controlling element " or " promoter " represents reflect nucleic acid one Point, the part generally (but not always) positioned at the upstream of the protein coding region of gene, the code area can include DNA or RNA, Or both DNA and RNA.When control region for it is active and with gene of interest be in operability combine in the state of or with When connecting to genetic manipulation of interest, this can realize the expression of gene of interest.Controlling element can mediate device Official's specificity, or control development or temporal gene activation." control region " can include promoter element, show basal promoter Activity core promoter element, have response in the inductivity of outside stimulus element, mediate promoter activity element, such as Negative regulatory element or transcriptional enhancer.As it is used herein, " control region " can also include element active after transcribing, example Such as, regulatory gene expression controlling element, such as translation and transcriptional enhancer, translation and Transcription inhibition, upstream activating sequence with And mRNA unstability determinant (mRNA instability determinants).Some in these latter elements can be with It is located adjacent to the position of code area.

In the context of this disclosure, term " controlling element " or " control region " are typically represented as generally (but non-total Be) positioned at structural gene upstream of coding sequence (5 ') DNA sequence dna, its by provide to RNA polymerase and/or transcription institute The identification of the other factors needed in specific site to start, to control the expression of code area.Included it will be appreciated, however, that being located at 3 ' of other nucleotide sequences or sequence in son may also contribute to the regulation and control of the expression to code area of interest.Offer pair The identification of RNA polymerase or other transcription factors is to ensure that the example in the controlling element of specific site Initiation is promoter Element.Most of but not all promoter in eukaryote element includes TATA boxes, includes adenosine and thymidine nucleotide base To conserved nucleic acid sequence (it is usually located at the base-pair of transcription initiation site upstream about 25).Promoter element, which includes, to be responsible for The basal promoter element of transcription initiation, and other controlling elements (such as listed above) of the modification of gene expression.

The control region of several type be present, include developmental regulation type, induction type or composing type those.Developmental regulation type Or control gene be in its differential expression under controlling special time of the control region during the development of the organ or tissue, It is activated in some organs or organ-tissue.However, the control region of some developmental regulation types is preferably in the specific stage of development Active in some organ or tissues, they can also be active in a manner of developmental regulation, or in plant Other organ or tissues in also exist with foundation level.The example in Tissue-specific regulatory area is (for example, see- specific regulatory controls Area) include napin promoters and cruciferin promoters (Rask et al., 1998, J.Plant Physiol.152:595- 599；Bilodeau et al., 1994, Plant Cell 14:125-130).The example of leaf specificity promoter includes plastocyanin Promoter (referring to US 7,125,978, it is incorporated herein by reference).

Inducible regulatory area is that response can directly or indirectly activate one or more DNA sequence dnas or base in derivant The control region of the transcription of cause.When in the absence of derivant, the DNA sequence dna or gene will not be transcribed.Typically, it is specific Being bound to inducible regulatory area can exist with the protein factor of activated transcription with inactive form, itself then to pass through derivant Directly or indirectly it is converted into activity form.However, the protein factor can also be not present.The derivant can be chemistry Reagent, such as protein, metabolite, growth regulator, herbicide or phenolic compound, or pass through hot, cold, salt or toxicity The physiological stress that element is direct acting or effect by pathogen or disease vector (such as virus) applies indirectly.It can pass through To the cell or plant external application derivant, and the plant cell containing inducible regulatory area is exposed to derivant, example Such as, by spraying, watering, heating or the like to carry out.Inducible regulatory element can derive from plant gene or non-plant Thing gene is (for example, Gatz, C.and Lenk, LR.P., 1998, Trends Plant Sci.3,352-358；It passes through reference It is incorporated to).The example of possible inducible promoter include but is not limited to tetracycline inducible promoter (Gatz, C., 1997, Ann.Rev.Plant Physiol.Plant Mol.Biol.48,89-108；It is incorporated by reference into), steroid inducible Promoter (Aoyama.T.and Chua, N.H., 1997, Plant 1.2,397-404；It is incorporated by reference into) and ethanol lure Conductivity type promoter (Salter, M.G. et al., 1998, Plant Journal 16,127-132；Caddick, M.X. et al., 1998, Nature Biotech.16,177-180, it is incorporated by reference into), the IB6 and the genes of CKI 1 of basic element of cell division induction (Brandstatter,I.and K.ieber,1.1.,1998,Plant Cell 10,1009-1019；Kakimoto,T., 1996,Science 274,982-985；It is incorporated by reference into) and growth hormone induction type element DR5 (Ulmasov, T. et al., 1997,Plant Cell 9,1963-1971；It is incorporated by reference into).

Composing type control region continuously instructs gene table in some of plant and in whole plant development process Reach.The example of known composing type controlling element includes the promoter related to following genes or transcript, the gene or turns Record and be originally：CaMV 35S transcripts (Odell et al., 1985, Nature, 313:810-812), (Zhang of rice actin 1 Et al., 1991, Plant Cell, 3:1155-1165), actin 2 (An et al., 1996, Plant J., 10:107-121) Or tms 2 (U.S.5,428,147, it is incorporated herein by reference), and triose phosphate isomerase 1 (Xu et al., 1994, Plant Physiol.106:459-467) gene, and the gene of maize ubiquitin 1 (Cornejo et al., 1993, Plant Mol.Biol.29:637-646), the gene of arabidopsis ubiquitin 1 and 6 (Holtorf et al., 1995, Plant Mol.Biol.29: 637-646), and tobacco translation initiation factor 4A genes (Mandel et al., 1995, Plant Mol.BioI.29:995- 1004)。

The gene that as used herein term " composing type " not necessarily shows to be under the control of composing type control region is in institute Have with identical horizontal expression in cell type, but mean that the gene is expressed in extensive cell type, although through It is frequently observed abundance difference.Composing type controlling element can operatively be connected with other coupling sequences with further enhancing them The transcription and/or translation of the nucleotide sequence connect.For example, CPMV-HT systemic origins are in non-the turning over of cowpea mosaic virus (CPMV) Region is translated, and shows the translation of related coding sequences enhancing." natural " represent that nucleic acid or amino acid sequence are naturally to deposit , or " wild type "." being operatively connected " represents particular sequence (for example, code area of interest and regulation and control member Part) directly or indirectly interaction, to play expected function, such as mediation or regulatory gene expression.The sequence being operatively connected The interaction of row can be mediated for example by the albumen to be interacted with the sequence being operatively connected.

The rotavirus VP 7 structural protein for including plant specificity N glycan can be induced in the RLP of plant endogenesis production Matter.Therefore, present invention also offers the RLP for including the VP7 with plant specificity N glycan.

In addition, the modification to N- glycan in plant be it is known (see, e.g. U.S.60/944,344；It passes through reference It is incorporated herein), and the VP7 with the N- glycan through modification can be produced.It can obtain and include the glycosylation pattern through modification VP7 (for example, N- glycan of fucosylation, xylosyl or fucosylation and both xylosyls with reduction), Or the VP7 with the type of glycosylation through modification can be obtained (wherein protein lacks fucosylation, xylosyl or two Person, and the galactosylation comprising raising).In addition, the regulation (for example, addition of terminal galactose) to posttranslational modification can To cause to decrease for the wild-type plant of the VP7 of expression fucosylation and xylosyl than expression VP7.

Such as (but being not construed as limiting), can by by VP7 and coding β -1.4 galactosyltransferases (GalT) The nucleotide sequence of (such as, but not limited to mammal GalT or people GalT, it is also possible to use the GalT in other sources) is together Coexpression, to realize the synthesis of the VP7 with the type of glycosylation through modification.GalT catalytic domain can also be fused to N- second The CTS domains (i.e. kytoplasm tail, membrane spaning domain, stem area) of acyl glucose aminopherase (GNT1) are miscellaneous to produce GNT1-GalT Enzyme is handed over, and the hybrid enzyme can co-express with VP7.VP7 can also be with encoding N- acetylglucosaminyl transferase III (GnT- III) (it is such as, but not limited to mammal GnT-III or people GnT-III, can also use the GnT- from other sources III nucleotide sequence) co-expresses together.Furthermore it is also possible to using GNT1-GnT-III hybrid enzymes, it includes and is fused to GnT- III GNT1 CTS.

Therefore, present invention also offers the RLP for including the VP7 with the N glycan through modification.

In the case where being not wishing to be bound by theory, the presence of the plant N- glycan on VP7 can be by promoting VP7 with resisting The combination of former presenting cell stimulates immune response.Saint-Jore-Dupas et al. (2007) has been presented for gathering using plant N Sugar stimulates immune response.

The present invention will be further described in the examples below.

Embodiment

Embodiment 1

The expression of rotavirus protein and VLP generation in Ben Saimushi tobaccos (N.benthamiana) plant leaf blade

Following analysis has used the Rotavirus capsid protein matter from G9P [6] rotavirus strain, and have evaluated class wheel Whether shape virion forms in multiple compartments of Ben Saimushi tobaccos (N.benthamiana) leaf cell.To tobacco plant The coexpression of VP2 and VP6 coexpression and VP2, VP6, VP7 and VP4 multiple combinations is studied in blade.

Material and method

Plasmid construction

For the rotavirus cDNA of VP2, VP4, VP6 and VP7 through vegetable codon optimization be by Geneart, What Germany was provided.According to the specification of manufacturer, plastid DNA is converted to DH5- α Competent Escherichia coli (E.coli) cell (E.cloni^TM,Lucigen).Use in our current research by Rainer Fischer (Fraunhofer Institute for Molecular Biology and Applied Ecology, IME, Germany) provide new two First agrobacterium vector pTRAc (cytoplasm), pTRAkc-rbcs1-cTP (targeting chloroplaset) and pTRAkc-ERH (targeting endoplasms Net).Another carrier pTRAkc-A (apoplast) derives to be digested by the restriction enzyme (RE) carried out in NcoI and XhoI sites In the modification (Fig. 3) that multiple cloning sites are carried out to pTRAkc-ERH.This eliminates histidine-tagged and KDEL sequences, and (they will Protein is retained in ER).Alternatively, the protein is targeted to apoplast.

Restriction enzyme (RE) digestion is carried out to VP2, VP4 and VP6 with NcoI/XhoI, and VP7 is cut with AflIII/XhoI.Limit Enzyme Afllll, NcoI and MluI processed have compatible cohesive end.In order to which DNA Direct Clonings are entered into pTRAc, pTRAkc-rbcs- CTP and pTRAkc-A, RE digestion is carried out to every kind of carrier in AflIII/XhoI, MluI/XhoI and NcoI/XhoI site respectively. The clone of DNA in carrier is carried out according to standard schedule, is then transformed into Competent Escherichia coli (E.coli) DH5- α cells (E.cloni^TM, Lucigen) in.Confirm selected restructuring bacterium colony by bacterium colony PCR.In order to be cloned in pTRAkc-ERH, Expanded by PCR and add NotI restriction enzyme sites to replace the terminator codon of each in four rotavirus cDNA.With table 1 The primer amplification cDNA of middle detailed description.PCR reaction conditions include：It is denatured 5 minutes at 95 DEG C, followed by 5 following circulations, The circulation is that 30 seconds are denatured at 95 DEG C, anneals 1 minute at 52 DEG C and extends 1.5 minutes at 72 DEG C.Carry out following institute Other 20 circulations shown：At 95 DEG C at 30 seconds, 57 DEG C at 1 minute, 72 DEG C 5 minutes at 1.5 minutes, and 72 DEG C.Then, press According to the specification of manufacturer, the fragment of amplification is cloned into pGEM-T-Easy (Promega).Conversion is in Competent large intestine Bacillus (E.coli) DH5- α (E.cloni^TM, Lucigen) in carry out.Then bacterium colony PCR is carried out on selected bacterium colony, to it Its three construct is also so carried out.

Table 1：Rotavirus cDNA primers for ER carrier clonings

PGEM-VP DNA from positive bacterium colony are sequenced, to verify PCR fidelity.Digested with NcoI/NotI DNA and appropriate DNA fragmentation is cloned into pTRAkc-ERH to form pTRAkc-ERH-VP in NcoI and NotI sites. Then, it is such as previously performed, carry out the conversion into Escherichia coli (E.coli) DH5- α cells.Also carried out bacterium colony PCR with Checked for the rotavirus DNA in selected bacterium colony.

Agrobacterium-mediated Transformation

Agrobacterium (Agrobacterium tumefaciens) GV3101 strains are taught by Rainer Fischer (Fraunhofer Institute for Molecular Biology and Applied Ecology IME,Aachen, Germany) provide, and become Electrocompetent (Shen and Forde, 1989) as mentioned before.In 0.1cm In electrogap cuvettes (BioRadTM), by the rotavirus pTRA-VP constructs and 100 μ l electroreceptions of 300ng separation State GV3101 mixing with cells, then with carry out electroporation arranged below in GenePulser (BioRad)：1.8Kv, 25 μ F and 200Ω.The incubation of 1 hour is carried out in 900 μ l LB at 27 DEG C, afterwards containing 50 μ g/ml carbenicillins (carb), Coated plate on the LA flat boards of 30 μ g/ml kanamycins (kan) and 50 μ g/ml rifampins (rif).Flat board is incubated 3 days at 27 DEG C.For Inspection positive transformant, from recombinational agrobacterium bacterium colony isolated plasmid dna, and it is converted back into Escherichia coli (E.coli) impression In state DH5- α cells.Then, they are screened 100 μ g/ml ampicillins (amp) LA are upper.Bacterium colony is carried out to cDNA PCR and limitation enzymic digestion are to confirm successful transformant.The glycerine for making related recombinational agrobacterium preserves strain, and by it in -70 DEG C storage.

Recombinational agrobacterium infiltrates

Agrobacterium (A.tumefeciens) LBA 4404 (pBIN-NSs) used in our current research derives from Marcel Prins(Laboratory of Virology,Wageningen University,Binnenhaven,Netherlands)。 It contains the NSs silencing suppressors found in tomato spotted wilf virus (TSWV).The recombinational agrobacterium of strain is preserved from glycerine (pTRA-VPs) grown overnight in the LB with 50 μ g/ml carb, 30 μ g/ml kan and 50 μ g/ml rif at 27 DEG C. Then, by recombinational agrobacterium with LBA4404 (pBIN-NSs) is every kind of is each inoculated in inducing culture (LB, 10mM 2- (N- Quinoline base) ethyl sulfonic acid MES, 2mM MgSO₄, 20 μM of acetosyringone, 50 μ g/ml carb, 30 μ g/ml kan and 50 μ g/ml Rif, and pH 5.6) in.

By culture in 27 DEG C of overnight incubations.Agrobatcerium cell is have collected by being centrifuged 5 minutes with 4000rpm at 4 DEG C, so 2ml infiltration mediums (10mM MES, 10mM MgCl is resuspended in afterwards₂, 3% sucrose, 5.6,200 μM of acetosyringones of pH And sterilized water) in.The optical density (OD600) of cell is demonstrated, and is diluted with infiltration medium to obtain 0.25 OD600.It is right For each pTRA-VP constructs, it is 0.5 that LBA4404 is mixed to final OD600 with recombinational agrobacterium.For coexpression Research, it is 0.5 that each construct, which is added to total OD600, for example, VP2-0.25 and VP6-0.25, until mixture OD600 etc. In 0.5.Contribute to the activation of vir genes in Agrobacterium in the acetosyringone for inducing with being used in infiltration medium.

Injured plant cell release phenolic compound, phenolic compound pass through the Vir A in Agrobacterium and Vir G bases Because being detected, it subsequently results in the induction (Zupan, J. et al., 2000) to protein expression in host cell.Then, will be thin Born of the same parents cultivate 1 hour at room temperature, to allow acetosyringone to induce vir genes.Soaked with the recombinational agrobacterium of expression VP protein Moisten wild type Ben Saimushi tobaccos (N.benthamiana) plant of three week old.This be related to the vacuum immersion of whole plant or Distal shaft the air gap recombinational agrobacterium (pTRA-VP) being injected on plant leaf blade veutro.With or without silencing suppressor LBA 4404 (pBIN-NSs) infiltrate to recombinational agrobacterium.

Initially, in a manner of each construct uses a syringe, 2ml agroinfiltration culture medium suspension is noted Inject each plant.In the experiment of timing in seven days, each construct uses one plant of plant.Rotavirus protein is also carried out Coexpression, wherein in the cytoplasm of Ben Saimushi tobaccos (N.benthamiana) plant leaf blade simultaneously express VP2, VP6 and VP4.The combination of combination and VP2/6/4 for VP2/6 is combined research.VP4 " furcella " protein can be bound to VP6, And therefore there is a possibility that they may be added to RLP structures.VP7 clones have been attempted, but have confirmed the toxicity in host cell Problem be present in aspect.Recombinate VP7 Agrobacteriums and kill leaf cell in one day after infiltration.Several method has been attempted to avoid Such case, such as infiltration of plants and being tested in timing is infiltrated after the 3rd day and/or the 5th day in a low temperature of 17 DEG C. Similarly, the poisonous property due to VP7 in tobacco plant, omitted in coexpression is studied.

Protein Extraction

Full leaf or two leaf dishes are harvested for each construct, and it is ground in liquid nitrogen.By the blade material of grinding It is resuspended in and includes adequate proteins enzyme inhibitor (no EDTA；Roche in sterile PBS).Then, centrifuged 5 minutes with 13000rpm, And discard precipitation group (plant leaf blade material).Then, 100 μ l each construct and 5 × SDS-PAGE loading buffer solutions are mixed Close, and boiled 2 minutes at 95 DEG C, prepare the further analysis for being carried out on PAGE gel and western blot.Will Remaining sample be stored in -20 DEG C it is standby.Fig. 4 shows rotavirus cDNA clone and the overview of infiltration flow.

Apoplast Protein Extraction

Extra extraction flow is carried out on apoplast construct pTRAkc-A.Apoplast be plant cell plasma membrane with it is thin Free diffusing space (Fig. 5 a) between cell wall.The protein expressed in cytoplasm has them targeted to apoplast Output sequence, therefore they accumulate in apoplast.In subsequent extraction flow, with the nothing for including adequate proteins enzyme inhibitor Bacterium PBS carries out vacuum or injection wetting to the full leaf from each extraction day.For vacuum immersion, single plant leaf blade is suspended In PBS, and placed 10 minutes in vacuum tank under 100mbar vacuum.Then, blade is rolled and put down gently in bottom (similar with Qiagen centrifugal columns) (Fig. 5 b2) in porose centrifugal column.The hole allows fluid easily from blade by without permitting Perhaps the blade material of solid passes through.Centrifugal column is placed in 2ml microcentrifugal tubes (Eppendorf tube), and with 4000rpm centrifuges 15 minutes (Fig. 5 b3).Collect filtrate and by the albumen for PAGE gel and Western blot analysis Matter loading dyestuff is added into every part of 100 μ l filtrate samples.

Western blot and coomassie dyeing

As it was noted above, using western blot and the PAGE gel through Coomassie blue stain.Printed in Western In mark, mouse anti-rotavirus VP6 antibody (US Biologicals) (1 is used:5000), the histidine-tagged antibody of anti-mouse ()(1:2000), the anti-VP2 of chicken and the anti-VP4 serum (1 of chicken:2000) protein of detection each respectively.Using examining horse The PAGE gel of this indigo plant dyeing, scanned by using the ribbon density of Syngene gel imaging systems, protein is carried out It is quantitative.

Electron microscope

In order to determine whether the protein of expression is assembled into RLP, in the case where silencing suppressor Nss be present, thin VP6, VP2/6 and VP2/6/4 of kytoplasm expression expression the 3rd day, has carried out the transmission electron microscope to arrested particles are immunized (TEM) analyze.By carbon/copper mesh of glow discharge in 20 μ l mouse anti-rotavirus VP6 antibody (1:5000) placed 5 minutes on, Then cleaned 3 times with sterile distilled water.Then, the net is placed on 10 μ l protein extracts, and retain 2 minutes, then Cleaned 3 times with sterile distilled water again.Finally, the net is floated 1 minute in the uranyl acetates of 20 μ l 2%, then in TEM Observed under (the OMEGA energy filtering projection electron microscopes of Zeiss 912, University of Cape Town).

For the sample separated from saccharose gradient, sucrose must be removed by dialysing first before trapping is immunized on copper mesh. If do not removed, because sucrose is forming crystal on the net, so as to destroy the structure with reference to carbon and material, sucrose crystal is caused to suppress sample Product qualitative observation really under the tem.Sucrose level is positioned in 10000MW dialysis cassettes, and in the nothing for including 0.4M NaCl Dialysed 4 hours in bacterium PBS, then change buffer solution, and it is stirred overnight in 4 DEG C.Because volume increases with dialysis, therefore Protein example needs to concentrate.By sample vacuum freeze drying 3 hours and it is resuspended in the sterile PBS of 2ml, prepares to be used for further Analysis.

RLP's is sucrose gradient purified

Initially, phytoprotein extract is filtered through micropore cloth, to remove solid plant matter.In each 40ml pipes, By producing six layers of 5ml sucrose for being dissolved in sterile PBS (pH 7.4), the saccharose gradient from 10 to 60% sucrose is established.So Afterwards, the protein example of clarification is loaded into the top of each gradient tubing string with 5 to 10ml volume.

At 4 DEG C, ultracentrifugation (SWTi28 swinging bucket rotors, the Beckman of 30 minutes 1 hour are carried out with 150 000g Coulter).At the end of centrifugation, punctured by pipe from each tubing string bottom collection 2ml fractions.Then, Dot blot point is carried out Analysis, to determine the fraction with protein of interest.For every kind of fraction, 1 μ l samples are loaded on nitrocellulose filter It is online, then it is closed with BSA Block buffers.Then, Western blot analysis is carried out in a conventional manner. With the anti-V6 antibody (1 of mouse:5000) protein VP6 is detected, or with the anti-VP2 of chicken and VP4 serum (1:5000) other two kinds are detected Protein.

Total soluble protein determines

Examined by Bradford, to determine total soluble protein (TSP).The inspection is carried out to compare in cytoplasm altogether The VP2/6 of expression protein accumulation is horizontal.Protein IgG (1.43mg/ml stostes) is used as standard to be serially diluted thing Product.5 μ l standard items and sample are added separately in clean drying microtiter plate.According to the specification of manufacturer (Bio-Rad Dc protein determinations), add total soluble protein reagent A and B.All experiments are repeated with three parts.Use Absorbance readings at ELIASA (Bio-tek PowerWave XS) record 750nm.

As a result

VP6 expression in leaves of plants cellular compartment

VP6 is expressed in the case where being with or without silencing suppressor, and is targetted all cellular compartment (Fig. 6；(line mark VP6 is shown, it is in about at 42kDa)).In cytoplasm, protein was expressed from being tested first day timing, at 1 week The accumulation of protein gradually steps up (Fig. 6 a) in cytoplasm during experiment.In ER, only protein was clearly observed at the 3rd day Accumulate (Fig. 6 b).The protein is shown with the stripe size (about have more 11kDa) higher than other oroteins.This is probably Caused by the 6- in the C-terminal of protein and cracking site (referring to pProEx carrier sequences) addition is histidine-tagged.

(Fig. 6 " chloroplaset ") occurs between the 1st day and the 3rd day for the protein accumulation in chloroplaset.Due to lack it is heavy Protein is can't detect in the case of silent repressor, therefore silencing suppressor has an impact to protein.Do not have the 5th day and the 7th day There is protein expression.Identical with ER, apoplast has optimal protein between the 3rd day to the 5th day that timing is tested Accumulate (Fig. 6 " apoplast "), and do not had completely then at the 1st and the 7th day.Silencing suppressor especially had positive role at the 3rd day, This causes higher protein detection compared with the situation of no silencing suppressor horizontal.It is also found that in about 40kDa mark Two band are observed that at note, this is probably due to caused by the signal label cutting on VP6 protein.

At the 3rd day, ER, chloroplaset and apoplast showed highest protein expression, wherein silence suppression be present The protein accumulated in the case of son is most.Due to the protein table for showing height in whole timing experiment and gradually stepping up Reach, therefore for protein accumulation, cytoplasm is optimal.

The expression of the rotavirus protein of histidine mark in cytoplasm

Four rotavirus vps are cloned into extra carrier (pTRAc-HT).The carrier includes targetting cytoplasmic egg The 6- of white matter is histidine-tagged, also, if the antibody of protein of interest can not be obtained, then by using anti-histidine mark The antibody of label enables to detection easily to carry out.In our case, only VP6 has commercially available antibody, therefore is waiting While treating serum, we have attempted all proteins the flow and have carried out early detection.Cytoplasm is same to VP6 expression suitable With, and promote us to attempt other oroteins.

The western blot result of the extract of the 3rd day is shown in the experiment of timing in 7 days：VP2, VP4 and VP6 successful expression (Fig. 7 a).In order to obtain VP7 expression in plant, multiple technologies have been attempted.However, opened with the VP7 plants infiltrated from the 1st day Begin to show yellow leaf, and progressed to wither (Fig. 7 b) during the process of timing experiment.It can't detect under these conditions Protein expression, also it can't detect after infiltrating the 1st day when plant still seems fairly good.

VP2 and VP4 expression in plant

VP2 and VP is infiltrated in Ben Saimushi tobaccos (N.benthamiana) plant leaf blade, and is targetted ER, Ye Lv Body, cytoplasm and apoplast.Because we can not obtain any positive colony in the Escherichia coli (E.coli), therefore our nothings The VP2 of method expression targeting apoplast carrier.However, the protein is targeted to all other 3 compartments (figure by successfully expression 8a).The anti-VP2 of chicken and anti-VP4 serum (1 have been used in the Western blot analysis to extract:2000).Respectively such as Fig. 8 a Shown in 8b, VP2 is visible at 100kDa marks (protein band indicated by an arrow) place immediately below with VP4 bands.For VP2, it appears that its expression in cytoplasm and ER is optimal, and for VP4, then it is optimal in cytoplasm and apoplast.It is heavy Silent repressor does not have remarkable effect to protein expression.As that can see from western blot, it is only in VP2ER structures Build in body and be slightly increased expression, it is still not so much in other.In the case where silencing suppressor be present, VP4 constructs are equal It is expressed.

VP2/6 and VP2/6/4 coexpression in cytoplasm

Cytoplasm is optimal for seeming expression to Rotavirus capsid protein matter, and shows highest extraction effect Rate.Therefore, all further expression work are completed with the cytoplasmic protein of targeting.

VP2 and VP6, which has been shown in, forms the RLP with protective immunity originality response in mouse, therefore, have studied thin VP2/6 and VP2/6/4 coexpression in kytoplasm.With anti-VP2 and anti-VP4 serum (1/5000) and the anti-VP6 antibody (1 of mouse： 5000) western blot is carried out, the VP2/6/4 of coexpression the 3rd day extract is detected (Fig. 9).It is such as previously determined , VP6 expression is very high, but the expression that VP2 and VP4 are can be seen that from the very fuzzy band of 100kDa marks is non- It is often low.This can be attributed to coexpression, and it causes to have used more host cell resources in VP6 overexpression, so as to be VP2 And/or the resource that VP4 leaves is less.If the band detected is any in both VP2 and VP4 or two kinds of protein Person, then and it is not easy what is determined.Obviously band on 130kDa is probably Dimerized VP6 protein. In the visible band of 55kDa marks most likely substantial amounts of phytoenzyme Rubisco.

VP6 to cytoplasmic expression and VP2/6 and VP2/6/4 to coexpression have carried out transmission electron microscope point Analysis, to check the RLP (Figure 10) of protein particulate and assembling.This further defines whether VP2 and/or VP4 successfully co-expresses really. When single expression, VP6 is assembled so as to form protein sheath, as shown in the arrow in Figure 10 b.When adding VP2, particle assembling Form RLP (Figure 10 c).VP2 plays the bone for enabling other oroteins to assemble and ultimately forming complete rotavirus structure The effect of frame protein.VP6 is also in conjunction with the VP4 structures to VP2, but still in being not easy the VP2/6/4 that determination co-expresses.Figure Electron micrograph in 10d is probably the VP2/6 particles assembled merely.But VP4 is combined during being shown in protein assembly To VP6, and this occurs before VP7 combinations.It is unstable and may be for electronics to be possible to these VP4 structures Depart from during the preparation flow of microscopy from RLP structures.

VP2/6's and VP2/6/4 is sucrose gradient purified

VP2/6 and VP2/6/4 (Figure 11 a) have been purified on the saccharose gradient of 10 to 60% sucrose.Collected from every bottom of the tube 2ml fractions, and detected with the anti-VP6 antibody of mouse and/or chicken anti-VP2 and VP4 serum, to determine to contain the protein Fraction.For VP2/6, protein is found that in fraction 16 and 17, because they are sun for VP6 albumen on trace (Figure 11 b) of property.In all fractions, the VP2/6/4 engram analysis carried out using the anti-VP2 of chicken and VP4 serum show sun Property result.This is probably due to caused by the high-level protein detection background by chicken serum.However, as seen in Figure 11 c , the spot intensity highest in fraction 17 and 18, this is probably that concentration is higher in these fractions due to protein of interest Caused by.

Summarize these results (Figure 11 b and Figure 11 c) and find that rotavirus protein is present in the scope of fraction 16 to 20 In.

Western blot and the coomassie dyeing of fraction

Western blot and SDS-PAGE are carried out to verify VP2 with VP6 protein in fraction 13 to the VP2/6 of coexpression Presence into 20.The Western blot analysis of the VP6 protein detected with the anti-VP6 antibody of mouse is in fraction 16 until in 20 It is positive (Figure 12 a, bottom arrow and Figure 12 c).The VP2 eggs detected with the anti-VP2 serum of chicken are detected in fraction 17 to 20 White matter (Figure 12 a, top arrow).In past coexpression research, VP2 has shown that expression is less than VP6, and this also schemes in the present invention Shown in 12a, wherein compared with VP6, the intensity of VP2 protein bands is relatively low.

On PAGE gel, the coexpression containing VP6 protein (Figure 11 b) is determined to previously passed Dot blot VP2/6 fraction 16 and 17 has carried out electrophoresis.By protein known to concentration, VP6 (the 0.91 μ g/ μ of SF9 insect cell expressions L) include, to determine the concentration of VP2/6 thick protein (Figure 11 b and Figure 11 c).This be by using Syngene gels into Carried out as system carries out density scan to crude protein band (swimming lane is labeled as crude protein), it is so that we can determine that The VP2/6 of every kilogram of blade material amount.It was found that Protein yield is about 1.54g/kg fresh weights (FW).From 1 gram of vegetable material Obtain the RLP (1.1g/kg) of 1.1mg purifying.

VP2/6 total soluble protein measure

Total soluble protein (TSP) is determined in the VP2/6 fractions of coexpression, to determine the relative of VP2/6 protein Measure (Figure 13).By using IgG standard items, the protein concentration of fraction 17 and 18 be respectively calculated as 0.538mg/ml and 1.012mg/ml (Figure 13 a).By the density scan on Syngene gel imaging systems to corresponding to VP2/ in these fractions 6 protein band is calculated, and it is respectively about 0.108mg/ml and 0.202mg/ml to find them.

Therefore, the TSP of the VP2/6 in fraction 17 and 18 is about 20%TSP.It was found that at most of RLP in sucrose post Between 15 to 25% sucrose, corresponding to fraction 15 to about fraction 20, emergent peak is now recorded on figure, is then disappeared Move back.The density variation of multiple material enables us to separate and thus purify protein of interest in extract.Use coomassie The PAGE gel of indigo plant dyeing only shows a significant band, and this represents that protein is relatively pure (Figure 12 b).

The VP2/6 of purifying TEM

The VP2/6 fractions of purifying are merged together, and dialysed in high salt PBS to remove sucrose, then saturating Penetrate on electron microscope and observe.Implement TEM to determine purity, and check whether RLP keeps completely after flow is purified.Such as figure Shown in 15, the major part in the background material being mainly made up of host cell product (Figure 10 b, c and d) is eliminated, is left RLP.Most of RLP keeps complete, but some seem to lose shape, and this is probably as the deformation caused by condition online EM It is caused.

The initial analysis that rotavirus structural protein matter is expressed in Ben Saimushi tobaccos (N.benthamiana) blade

The initial analysis focuses on Ben Saimushi tobaccos (N.benthamiana) blade (as exemplary host expresses System) in rotavirus structural protein matter VP2 (SEQ ID NO：1)、VP4(SEQ ID NO：2)、VP6(SEQ ID NO：3) With VP7 (SEQ ID NO：4) expression.Rotavirus strain selected by the present invention is Major Epidemic in South Africa and other spreads Kazakhstan Draw G9P [6] strain of areas to the south.Target this strain RLP vaccines will be helpful to mitigate the Sahara on the south African disease bear Load.

Use in this analysis by agriculture bacillus mediated transient expression system.With transgene expression on the contrary, instantaneous table Up to allowing protein quickly to be expressed within the relatively short time, wherein Rotavirus capsid protein matter gene unconformity contaminates in host In colour solid.Most of protein is expressed, and in recombinational agrobacterium infiltration Ben Saimushi tobaccos (N.benthamiana) blade Detectable amount is built up at the 3rd day.It is as follows, several rotavirus structure is observed in leaves of plants cellular compartment The successful expression of property protein, including VP2, VP4 and VP6, as described in detail in table 2：

Table 2：Rotavirus vp protein expression in a variety of leaf cell compartments

0=is expressed without expression 1=

Not it was observed that glycoprotein VP7 expression, this may be due to caused by its toxic action to plant cell.It is worth note Meaning, is used for the Primary Study by the VP7 comprising its natural signals peptide.It has also been attempted and co-expressing the during testing the 3rd It is infiltrated.Carry out this trial and come whether observing protein is expressed and whether then assembled soon with VP2 and VP6 To form RLP.In our current research it was observed that restructuring VP7 toxicity properties be described before this (Williams et al., 1995；McCorquodale, 1987；Arias et al., 1986).

Have reported in transgenic potato VP7 expression studies (Li et al., 2006；Choi et al., 2005； Wu et al., 2003).Choi et al. (2005) uses ape rotavirus VP 7, and Li et al. and Wu et al. (Li et al., 2006；Wu Et al., 2003) user's A groups G1VP7.Result as described herein has used human rotavirus G9VP7.

VP2 is expressed and is targetted all compartments in addition to apoplast, because we can not clone suitable cDNA, And the time, which limits, only allows us to be attempted several times before being carried out using other constructs.Notice VP2 expression water Put down significantly lower in all compartments.In the past research quoted in Saldana et al., 2006, obtain to draw a conclusion：To the greatest extent Pipe detects mRNA in plant cell, but the VP2 that its sequence optimized for the expression in plant can not possibly be expressed.But he Successfully expressed in tomato plant cell using synthetic DNA.The reason for VP2 expression is difficult is likely due to not Correct mRNA translations or mRNA, which are included, makes (Kawaguchi and caused by the unstable some sequence motifs of plant cell Bailey-Serres,2002).The evidence horizontal than VP2 low expressions for VP6 seen Mena et al. (2006), The plant of Saldana et al. (2006), Vieira et al. (2005) and Labb é et al. (1991) and insect cell expression are studied In.

The outer capsid proteins matter VP4 that furcella is formed on virus particle structure surface is expressed and targetted, with cytoplasm, ER in apoplast with accumulating.Protein accumulation is not detected by chloroplaset.As observed by being directed to VP2, western blot On, VP4 protein expression level is less than seen by VP6.The protein has positioned trypsin cleavage site, and this will be produced Two kinds of protein：VP5 and VP8.It is possible to following situations occur：Office in Ben Saimushi tobaccos (N.benthamiana) blade Portion's trypsase cuts some protein when they are produced, so as to cause the complete VP4 accumulated in compartment is specified dense Degree is horizontal relatively low.The protein has been shown as main neutralization antigen, but some existing trials are used for clone holoprotein Vaccine development (Khodabandehloo et al., 2009；Mahajan et al., 1995；Nishikawa et al., 1989).However, Insect cell has shown expression (Andr é s etc. of VP4 VP5 or VP8 subunits with some researchs in yeast expression system People, 2006；Favacho et al., 2006；Kovacs-Nolan et al., 2001).Up to the present, this research is display plant The first term research that holoprotein is expressed in expression system.

VP6 is expressed in all compartments, is overexpressed wherein being observed in cytoplasm, from the 1st day to the 7th in the compartment It observes protein accumulation.This is with pointing out proteinase activity and gene silencing by extraneous protein in reduction or block cell matter Accumulation some documents it is opposite (Fischer et al., 2004).In addition, it is contemplated that correct pH conditions, it is known that VP6 self assemblies Into tubulose or spiral particle, like the particle (Fig. 9 b) (Estes et al., 1987) observed in our study.VP6 accounts for disease About the 50% of malicious core, and be therefore the major antigen of Rotavirus Vaccine exploitation.The result that the above is obtained enables us VP2, VP6 and VP4 coexpression in enough further research cytoplasm.

When being co-expressed in cytoplasm, VP2 and VP6 are assembled to form RLP.It was observed that from transient expression system 1.27-1.54g/kg the very high protein output between FW.When being purified on sucrose post, resident VP amount is 1.1g/kg FW.IgG antibody of the yield with using transient expression system in Ben Saimushi tobaccos (N.benthamiana) It is suitable to produce the up to 1.5g/kg FW yield (V é zina et al., 2009) obtained.Saldana et al. (2006) be to It is the only known so far rotavirus vp 2 and VP6 successfully to be co-expressed in Transgenic Tomato Plants and to reach about 1% total The horizontal seminar of soluble protein.VP2/6 assembling records (Vieira etc. by detailed in insect cell expression system People, 2005；O ' Brien et al., 2000).These VP2/6RLP, which have also been shown, provides protective immunity to keep out colyliform disease Poison infection (Zhou et al., 2011；Saldana et al., 2006).Therefore, our caused VP2/ in plant expression system 6RLP is the suitable candidate for developing subunit's Rotavirus Vaccine.

VP2/6/4 is also co-expressed and detected.First in the gross protein absorbance readings of the protein of coexpression Individual visible peak (Figure 14, fraction 16) is probably the VP2/6/4 of assembling, but observes this grade of timesharing under the tem and be not detected by RLP.See The protein peak observed can be as caused by VP4 monomers or its respective VP5 with the accumulation of VP8 subunits.When examining under the tem When looking into, second peak (fraction 18) shows the RLP structures of those for being very similar to be observed in VP2/6 samples.However, Crawford et al. has previously been reported can not observe VP4 under the tem, and VP2/6/4 and VP2/6/4/7 particles under the tem With similar structures and diameter (Crawford 1994).We enter to VP2/6/7RLP, VP 2/6/4/7RLP and VP2/6RLP Identical observation is gone, they seem entirely similar under conventional TEM.

Embodiment 2

Construct

A-2X35S/CPMV-HT/RVA (WA) VP2 (opt)/NOS (construct numbering 1710)

Using the method for following PCR-baseds, will encode the optimized sequence of the VP2 from rotavirus A WA strains comprising It is cloned into the plasmid of Plasto_pro/P19/Plasto_ter expression cassettes in 2X35S-CPMV-HT-NOS expression systems.Use Optimized VP2 gene orders (Figure 19, SEQ ID NO：45) template is used as, uses primer I F-WA_VP2 (opt) .s1+3c (Figure 17 A, SEQ ID NO：And IF-WA_VP2 (opt) .s1-4r (Figure 17 B, SEQ ID NO 21)：22) amplification encodes comprising VP2 The fragment of sequence.On sequence optimisation, by VP2 protein sequences (Genbank accession number CAA33074) reverse translation (backtranslate), and for people's codon using, G/C content and mRNA structures it is optimized.Use In-Fusion Cloning system (Clontech, Mountain View, CA), 2X35S/CPMV-HT/NOS expression systems are cloned into by PCR primer In.Construct numbering 1191 (Figure 17 C) is digested with SacII and StuI restriction enzymes, and the plasmid of linearisation is used for In Fusion assembling reactions.Construct numbering 1191 is intended to for " the In Fusion " in the expression cassette based on CPMV-HT Clone the receptor plasmid of gene of interest.It is also introduced for common under clover plastocyanin gene promoter and terminator Express the gene construct of TBSV P19 silencing suppressors.Main chain is pCAMBIA binary plasmids, and Figure 18 is shown from left t- The sequence (the SEQ ID NO on DNA borders to right t-DNA borders：23).Gained construct is numbered as 1710 (Figure 23, SEQ ID NO：27).Figure 20 shows amino acid sequence (the SEQ ID NO of the VP2 from rotavirus A strains WA：25).Figure 21 is shown The schematic diagram of plasmid 1710.

B-2X35S/CPMV-HT/RVA (WA) VP2 (opt)/NOS to BeYDV (m)+replicase amplification system (compile by construct Number 1711)

Using the method for following PCR-baseds, the optimized sequence for encoding the VP2 from rotavirus A WA strains is being wrapped It is cloned into the plasmid of the expression cassette containing Plasto_pro/P19/Plasto_ter comprising BeYDV (m)+replicase amplification system In 2X35S/CPMV-HT/NOS.Use optimized VP2 gene orders (SEQ ID NO：45) template is used as, uses primer I F- WA_VP2 (opt) .s1+3c (Figure 17 A, SEQ ID NO：And IF-WA_VP2 (opt) .s1-4r (Figure 17 B, SEQ ID NO 21)： 22) amplification includes the fragment of VP2 coded sequences.On sequence optimisation, by VP2 protein sequence (Genbank accession numbers CAA33074) reverse translation, and be optimized for people's codon using, G/C content and mRNA structures.Use In- Fusion cloning systems (Clontech, Mountain View, CA), by PCR primer in 2X35S/CPMV-HT/NOS expression cassettes In be cloned into BeYDV (m) amplification systems.Construct 193 (Figure 22 A) is digested with SacII and StuI restriction enzymes, and The plasmid of linearisation is used for In Fusion assembling reactions.Construct numbering 193 be intended to for by gene of interest in base " In Fusion " are cloned into the receptor plasmid in BeYDV (m) amplification systems in CPMV-HT expression cassette.It also introduces use In the gene construct that TBSV P19 silencing suppressors are co-expressed under clover plastocyanin gene promoter and terminator.Main chain For pCAMBIA binary plasmids, and Figure 22 B show the sequence (the SEQ ID from left t-DNA borders to right t-DNA borders NO：26).Gained construct is numbered as 1711 (Figure 23, SEQ ID NO：27).Figure 20 is shown from rotavirus A strains WA VP2 amino acid sequence (SEQ ID NO：25).Figure 24 shows the schematic diagram of plasmid 1711.

C-2X35S/CPMV-HT/RVA (WA) VP6 (opt)/NOS (construct numbering 1713)

Using the method for following PCR-baseds, will encode the optimized sequence of the VP6 from rotavirus A WA strains comprising It is cloned into the plasmid of Plasto_pro/P19/Plasto_ter expression cassettes in 2X35S-CPMV-HT-NOS expression systems.Use Optimized VP6 gene orders (SEQ ID NO：46) be used as template, using primer I F-WA_VP6 (opt) .s1+3c (Figure 25 a, SEQ ID NO：And IF-WA_VP6 (opt) .s1-4r (Figure 25 b, SEQ ID NO 28)：29) amplification includes VP6 coded sequences Fragment.On sequence optimisation, by VP6 protein sequences (Genbank accession number AAA47311) reverse translation, and it is close to be directed to people Numeral is optimized using, G/C content and mRNA structures.Use In-Fusion cloning systems (Clontech, Mountain View, CA), PCR primer is cloned into 2X35S/CPMV-HT/NOS expression systems.With SacII and StuI restriction enzymes to structure Build body numbering 1191 (Figure 17 C) to be digested, and the plasmid of linearisation is used for In Fusion assembling reactions.Construct is compiled Numbers 1191 are intended to for " In Fusion " to clone the receptor plasmid of gene of interest in the expression cassette based on CPMV-HT. It also introduces the base for co-expressing TBSV P19 silencing suppressors under clover plastocyanin gene promoter and terminator Because of construct.Main chain is pCAMBIA binary plasmids, and Figure 18 shows the sequence from left t-DNA borders to right t-DNA borders Arrange (SEQ ID NO：23).Gained construct numbering is 1713 (Figure 25 c, SEQ ID NO：30).Figure 26 is shown from colyliform Viral A strains WA VP6 amino acid sequence (SEQ ID NO：31).Figure 27 shows the schematic diagram of plasmid 1713.

D-2X35S/CPMV-HT/RVA (WA) VP6 (opt)/NOS to BeYDV (m)+replicase amplification system (compile by construct Number 1714)

Using the method for following PCR-baseds, will encode the optimized sequence of the VP6 from rotavirus A WA strains comprising It is cloned into the plasmid of Plasto_pro/P19/Plasto_ter expression cassettes comprising BeYDV (m)+replicase amplification system In 2X35S/CPMV-HT/NOS.Use optimized VP6 gene orders (SEQ ID NO：46) template is used as, uses primer I F- WA_VP6 (opt) .s1+3c (Figure 25 a, SEQ ID NO：And IF-WA_VP6 (opt) .s1-4r (Figure 25 b, SEQ ID NO 28)： 29) amplification includes the fragment of VP6 coded sequences.On sequence optimisation, by VP6 protein sequence (Genbank accession numbers AAA47311) reverse translation, and be optimized for people's codon using, G/C content and mRNA structures.Use In- Fusion cloning systems (Clontech, Mountain View, CA), by PCR primer in 2X35S/CPMV-HT/NOS expression cassettes In be cloned into BeYDV (m) amplification systems.Construct 193 (Figure 22 A) is digested with SacII and StuI restriction enzymes, and The plasmid of linearisation is used for In Fusion assembling reactions.Construct numbering 193 be intended to for by gene of interest in base " In Fusion " are cloned into the receptor plasmid in BeYDV (m) amplification systems in CPMV-HT expression cassette.It also introduces use In the gene construct that TBSV P19 silencing suppressors are co-expressed under clover plastocyanin gene promoter and terminator.Main chain For pCAMBIA binary plasmids, and Figure 22 B show the sequence (the SEQ ID from left t-DNA borders to right t-DNA borders NO：26).Gained construct numbering is 1714 (Figure 28, SEQ ID NO：32).Figure 26 is shown from rotavirus A strains WA's VP6 amino acid sequence (SEQ ID NO：31).Figure 29 shows the schematic diagram of plasmid 1714.

C-2X35S/CPMV-HT/RVA (Rtx) VP4 (opt)/NOS (construct numbering 1730)

Using the method for following PCR-baseds, coding is come from into rotavirus A vaccines USA/Rotarix-A41CB052A/ The VP4 of 1988/G1P1A [8] strain optimized sequence is in the plasmid comprising Plasto_pro/P19/Plasto_ter expression cassettes It is cloned into 2X35S/CPMV-HT/NOS.Use optimized VP4 gene orders (Figure 31 B, SEQ ID NO：47) it is used as mould Plate, use primer I F-Rtx_VP4 (opt) .s1+3c (Figure 30 A, SEQ ID NO：And IF-Rtx_VP4 (opt) .s1-4r 33) (Figure 30 B, SEQ ID NO：34) amplification includes the fragment of VP4 coded sequences.On sequence optimisation, by VP4 protein sequences (Genbank accession number AEX30660) reverse translation, and carried out for people's codon use, G/C content and mRNA structures excellent Change.Using In-Fusion cloning systems (Clontech, Mountain View, CA), PCR primer is cloned into 2X35S/ In CPMV-HT/NOS expression cassettes.With SacII and StuI restriction enzymes to (Figure 18, SEQ the ID NO of construct numbering 1191：23) enter Digestion is gone, and the plasmid of linearisation is used for In Fusion assembling reactions.Construct numbering 1191 be intended to be used for based on " In Fusion " clone the receptor plasmid of gene of interest in CPMV-HT expression cassette.It is also introduced in clover matter Body indigo plant plain gene promoter and the gene construct that TBSV P19 silencing suppressors are co-expressed under terminator.Main chain is pCAMBIA Binary plasmid, and Figure 18 shows the sequence (the SEQ ID NO from left t-DNA borders to right t-DNA borders：23).Gained Construct numbering is 1730 (Figure 31 C, SEQ ID NO：50).Figure 32 is shown from rotavirus A vaccines USA/Rotarix- A41CB052A/1988/G1P1A [8] VP4 amino acid sequence (SEQ ID NO：36).Figure 33 A show plasmid 1730 Schematic diagram.

E-2X35S/CPMV-HT/RVA (Rtx) VP4 (opt)/NOS to BeYDV (m)+replicase amplification system (construct Numbering 1731)

Using the method for following PCR-baseds, coding is come from into rotavirus A vaccines USA/Rotarix-A41CB052A/ The VP4 of 1988/G1P1A [8] strain optimized sequence is in the plasmid comprising Plasto_pro/P19/Plasto_ter expression cassettes It is cloned into the 2X35S/CPMV-HT/NOS comprising BeYDV (m)+replicase expression system.Use optimized VP4 gene sequences Arrange (SEQ ID NO：47) template is used as, uses primer I F-Rtx_VP4 (opt) .s1+3c (Figure 30 A, SEQ ID NO：33) and IF-Rtx_VP4 (opt) .s1-4r (Figure 30 B, SEQ ID NO：34) amplification includes the fragment of VP4 coded sequences.It is excellent on sequence Change, by VP4 protein sequences (Genbank accession number AEX30660) reverse translation, and for people's codon use, G/C content It is optimized with mRNA structures.Using In-Fusion cloning systems (Clontech, Mountain View, CA), PCR is produced Thing is cloned into 2X35S/CPMV-HT/NOS expression cassettes in BeYDV (m) amplification systems.With SacII and StuI restriction enzymes to structure Build body 193 (Figure 22 A) to be digested, and the plasmid of linearisation is used for In Fusion assembling reactions.Construct numbering 193 It is intended to for by gene of interest, " In Fusion " to be cloned into BeYDV (m) amplifications in the expression cassette based on CPMV-HT The receptor plasmid of system.It is also introduced is used for the suppression of TBSV P19 silences under clover plastocyanin gene promoter and terminator The gene construct of the coexpression of system.Main chain is pCAMBIA binary plasmids, and Figure 22 B show from left t-DNA borders to The sequence (the SEQ ID NO on right t-DNA borders：26).Gained construct numbering is 1731 (Figure 31, SEQ ID NO：35).Figure 32 show the amino acid sequence of the VP4 from rotavirus A vaccines USA/Rotarix-A41CB052A/1988/G1P1A [8] (SEQ ID NO：36).Figure 33 B show the schematic diagram of plasmid 1731.

F-2X35S/CPMV-HT/RVA (Rtx) VP7 (opt)/NOS (construct numbering 1733)

Using the method for following PCR-baseds, coding is come from into rotavirus A vaccines USA/Rotarix-A41CB052A/ The VP7 with its natural signals peptide of 1988/G1P1A [8] strain optimized sequence is including Plasto_pro/P19/ It is cloned into the plasmid of Plasto_ter expression cassettes in 2X35S-CPMV-HT-NOS expression systems.Use optimized VP7 genes Sequence (SEQ ID NO：54) template is used as, uses primer I F-Rtx_VP7 (opt) .s1+3c (Figure 34 A, SEQ ID NO：37) With IF-Rtx_VP7 (opt) .s1-4r (Figure 34 B, SEQ ID NO：38) amplification includes the fragment of VP7 coded sequences.On sequence Optimization, contain by VP7 protein sequences (Genbank accession number AEX30682) reverse translation, and for people's codon use, GC Amount and mRNA structures are optimized.Using In-Fusion cloning systems (Clontech, Mountain View, CA), by PCR Product cloning is into 2X35S/CPMV-HT/NOS expression systems.With SacII and StuI restriction enzymes to the (figure of construct numbering 1191 17C) digested, and linearization plasmid is used for In Fusion assembling reactions.Construct numbering 1191 is intended to be used for " In Fusion " clone the receptor plasmid of gene of interest in the expression cassette based on CPMV-HT.It, which is also introduced, is used for Clover plastocyanin gene promoter and the gene construct that TBSV P19 silencing suppressors are co-expressed under terminator.Main chain is PCAMBIA binary plasmids, and Figure 18 shows the sequence (the SEQ ID NO from left t-DNA borders to right t-DNA borders： 23).Gained construct numbering is 1733 (Figure 34 C, SEQ ID NO：24).Figure 35 is shown from rotavirus A vaccines USA/ The VP7 with natural signals peptide of Rotarix-A41CB052A/1988/G1P1A [8] strain amino acid sequence (SEQ ID NO： 39).Figure 36 shows the schematic diagram of plasmid 1733.

D-2X35S/CPMV-HT/TrSp-RVA (Rtx) VP7 (opt)/NOS (construct numbering 1734)

Using the method for following PCR-baseds, coding is come from into rotavirus A vaccines USA/Rotarix-A41CB052A/ The VP7 of the natural signals peptide with clipped form of 1988/G1P1A [8] strain optimized sequence is including Plasto_pro/ It is cloned into the plasmid of P19/Plasto_ter expression cassettes in 2X35S-CPMV-HT-NOS expression systems.Use optimized VP7 Gene order (nt 88-981, the SEQ ID NO corresponded in Figure 44 C：57) template is used as, uses primer I F-TrSP+Rtx_ VP7 (opt) .s1+3c (Figure 44 A, SEQ ID NO：And IF-Rtx_VP7 (opt) .s1-4r (Figure 44 B, SEQ ID NO 55)：56) Amplification includes the fragment of VP7 coded sequences.On sequence optimisation, by VP7 protein sequences (Genbank accession number AEX30682) Reverse translation, and be optimized for people's codon using, G/C content and mRNA structures.Cloned using In-Fusion and be Unite (Clontech, Mountain View, CA), PCR primer is cloned into 2X35S/CPMV-HT/NOS expression systems.With SacII and StuI restriction enzymes are digested to construct numbering 1191 (Figure 17 C), and the plasmid of linearisation is used for into In Fusion assembling reactions.Construct numbering 1191 is intended to for " In Fusion " to be cloned in the expression cassette based on CPMV-HT The receptor plasmid of gene of interest.It, which is also introduced, is used in clover plastocyanin gene promoter with being co-expressed under terminator The gene construct of TBSV P19 silencing suppressors.Main chain is pCAMBIA binary plasmids, and Figure 18 is shown from left t-DNA The sequence (the SEQ ID NO on border to right t-DNA borders：23).Gained construct numbering is 1734 (Figure 44 D, SEQ ID NO： 58).Figure 44 E show that carrying from rotavirus A vaccines USA/Rotarix-A41CB052A/1988/G1P1A [8] strain is cut The VP7 of short signal peptide amino acid sequence (SEQ ID NO：59).Figure 44 F show the schematic diagram of plasmid 1734.

G-2X35S/CPMV-HT/PDISP/RVA (WA) VP7 (opt)/NOS to BeYDV (m)+replicase amplification system (structure Build body numbering 1735)

Using the method for following PCR-baseds, coding is come from into rotavirus A vaccines USA/Rotarix-A41CB052A/ The VP7 of 1988/G1P1A [8] strain sequence is cloned into the plasmid comprising Plasto_pro/P19/Plasto_ter expression cassettes In 2X35S-CPMV-HT-PDISP-NOS expression systems.Use optimized VP7 gene orders (SEQ ID NO：54) it is used as mould Plate, use primer I F-Rtx_VP7 (opt) .s2+4c (Figure 37 A, SEQ ID NO：And IF-Rtx_VP7 (opt) .s1-4r 40) (Figure 34 B, SEQ ID NO：38) amplification includes the fragment of the VP7 coded sequences without his wild type signal peptides.On sequence Optimization, contain by VP7 protein sequences (Genbank accession number AEX30682) reverse translation, and for people's codon use, GC Amount and mRNA structures are optimized.Using In-Fusion cloning systems (Clontech, Mountain View, CA), by PCR Product is cloned into 2X35S/CPMV-HT/NOS expression cassettes in a manner of meeting reading frame with clover PDI signal peptides.Use SacII Construct 1192 (Figure 38) is digested with StuI restriction enzymes, and the plasmid of linearisation is used for In Fusion assemblings instead Should.Construct numbering 1192 is intended to for believing gene of interest with clover PDI in the expression cassette based on CPMV-HT Number peptide meets the mode " receptor plasmid of In Fusion " clones of reading frame.It is also introduced in clover plastocyanin base Because co-expressing the gene construct of TBSV P19 silencing suppressors under promoter and terminator.Main chain is pCAMBIA binary plasmids, And Figure 39 shows t-DNA borders (the SEQ ID NO of the sequence from left to right：41).Gained construct numbering is 1735 (figures 40, SEQ ID NO：42).Figure 41 is shown from rotavirus A vaccines USA/Rotarix-A41CB052A/1988/G1P1A [8] PDISP/VP7 of strain amino acid sequence (SEQ ID NO：43).Figure 42 shows the schematic diagram of plasmid 1735.

Description of the table 3. to the synthetic gene for producing RLP.

What the sequence * optimized was modified to be advantageous to preferable people's codon uses and improves G/C content.

Table 4. for RLP to producing and the description of assembled and test construct

WtSp：Wild type signal peptide, SpPDI：The signal peptide of plant origin, clone from property of alfalfa protein disulfide bond isomery Enzyme gene, TrSp：The wild type signal peptide of truncation, TrSp originate from second Met (M30) in WtSp.

* [optimization] represents that sequence is optimized using, G/C content and RNA structures based on codon.

Embodiment 3

The assembling of gene construct and Agrobacterium-mediated Transformation

All plasmids (including plasmid 1710,1713,1730 and 1734) are used to pass through electroporation (Mattanovich etc. People, 1989, Nucleic Acid Res.17:6747) Agrobacterium (Agrobacterium tumefaciens) (AGL1 is converted； ATCC, Manassas, VA 20108, USA), or alternatively, the competent cell prepared by CaCl2 can be used to carry out heat Swash (XU et al., 2008, Plant Methods 4).By drawing restriction map (restriction mapping), confirm The integrality of plasmid in caused Agrobacterium (A.tumefaciens) bacterial strain.The agriculture that will be converted through given binary plasmid Bacillus (A.tumefaciens) Strain Designation is AGL1/ " plasmid number ".For example, the agriculture bar that constructed body numbering 1710 converts Bacterium (A.tumefaciens) bacterial strain is named as " AGL1/1710 ".

Preparation, inoculation, agroinfiltration and the harvest of plant biomass

In the level land for filling commercially available bog moss matrix, from cultivating seeds Ben Saimushi tobaccos (Nicotiana Benthamiana) plant.Make plant under 16/8 periodicity of illumination and daytime 25 DEG C/night, 20 DEG C of temperature scenario in greenhouse Growth.After planting 3 weeks, single plant is picked, is transplanted in basin, and make its regrowth 3 in greenhouse under same environmental conditions Week.

It is being supplemented with the plant origin of 10mM 2- (N- morpholines) ethyl sulfonic acid (MES) and 50 μ g/ml kanamycins pH 5.6 The Agrobacterium that culture transfects through every kind of construct in LB culture mediums, until they reach the OD600 between 0.6 to 2.5.By agriculture bar Bacterium suspension mixes, and to reach appropriate ratio for every kind of construct, and uses infiltration medium (10mM MgCl₂And 10mM MES pH 5.6) become 2.5 × OD600.By Agrobacterium (A.tumefaciens) suspension in 4 DEG C of storage over night.Infiltration On the same day, batch cultivation thing is diluted with 2.5 times of suspension volumes with infiltration medium, and it is warmed before use.By Ben Saimu Bacterium of family name tobacco (N.benthamiana) whole plant under 20-40Torr vacuum in airtight stainless cylinder of steel is suspended It is inverted 2 minutes in liquid.After infiltration, plant is sent back to greenhouse, a period of time of culture 3-12 days is until harvest.By the biology of harvest Matter remains freezing state (- 80 DEG C), until the purifying for particle.

The extraction and purifying of class rotaviral particles

With the Extraction buffer (TNC of 3 times of volumes：10mM Tris pH 7.4、140mM NaCl、10mM CaCl₂) mixing Protein is extracted from the biomass of freezing by mechanicalness extraction in clutch.By slurries filter through macropore nylon filter, with except Big fragment is removed, and is centrifuged 5 minutes with 5000g at 4 DEG C.Collect supernatant and again with 5000g centrifugation 30 minutes (4 DEG C), with except Remove other fragments.20 minutes (4 DEG C) are centrifuged with 75000g by supernatant depth-type filtration and ultrafiltration, and by filtrate, to concentrate class Rotaviral particles.Particle comprising particle is resuspended in the TNC of 1/12 times of volume, centrifuges 5 minutes by 5000g to remove Remove insoluble matter.The filtering supernatant on micropore cloth, it is loaded into afterwards in Iodixanol density gradient.

It is as follows, carry out density gradient centrifugation.The pipe for including the stagewise gradient from 5% to 45% Iodixanol is prepared, And covered with the filtrated extract comprising class rotaviral particles.The gradient is centrifuged into 4 hours (4 DEG C) with 120000g.Centrifuging Afterwards, the SDS-PAGE and western blot dyed from bottom to collected overhead 1ml fractions and by coomassie is analyzed.For The Iodixanol of fraction that selection is used to further analyze is removed, selected fraction is centrifuged into 20 minutes (4 DEG C) with 75000g, and And the particle of precipitation is resuspended in fresh TNC buffer solutions.

SDS-PAGE and immunoblotting assay

Protein concentration is determined by BCA protein assays (Pierce Biochemicals, Rockport IL).Reducing Or by SDS-PAGE protein isolates matter and use Coomassie blue stain under non reducing conditions.Scan the gel of dyeing and use ImageJ softwares (NIH) carry out spectrodensitometry analysis.

For immunoblotting assay, by the albumen electrotransfer after electrophoresis to poly- difluoroethylene (PVDF) film (Roche Diagnostics Corporation, Indianapolis, IN) on.Before immunoblotting assay, delayed in 4 DEG C using Tris 5% skimmed milk and 0.1% Tween-20 in fliud flushing (TBS-T) carry out the closing of 16-18 hours to film.

By the way that suitable antibody (table 5) is incubated in 2% skimmed milk in TBS- polysorbas20s 0.1% to enter with 2 μ g/ml Row immunoblotting assay.Each secondary antibody for chemiluminescence detection is as shown in table 5, as shown in TBS- tweens They are diluted in 2% skimmed milk in 200.1%.Use luminol (Roche Diagnostics Corporation substrate) is used as, passes through chemiluminescence detection immunocompetence compound.By using EZ-LinkIt is living Horseradish peroxidase-the enzyme for changing peroxidase conjugated kit (Pierce, Rockford, IL) progress human IgG antibody is sewed Close.

Table 5：Deposition condition, antibody and the dilution factor of the immunoblotting assay of wheel virus antigen.

Anti- VP4 enzyme linked immunosorbent assay (ELISA)s (ELISA)

With 1 in 10mM PBS pH7.4 (phosphate buffered saline (PBS)), 150mM NaCl:100000 times dilute it is small The anti-VP4 of murine monoclonal (being provided by Koki Taniguchi professor's good wills) is in 4 DEG C to the hole microtiter plate coating 16- of U-shaped bottom 96 18 hours.After incubation, with the 10mM PBS pH7.4 comprising 0.1% Tween-20,1M NaCl clean plates three times, and with comprising 5%BSA in 10mM PBS pH 7.4, the 150mM NaCl of 0.1% Tween-20 carries out the closing of 1 hour in 37 DEG C to plate. After step is closed, with the 10mM PBS pH 7.4 comprising 0.1% Tween-20,1M NaCl clean plates three times.Add sample Product, and plate is incubated 1 hour in 37 DEG C.Then, with the 10mM PBS pH 7.4 comprising 0.1% Tween-20,1M NaCl, 1mM CaCl₂、0.5mM MgCl₂Clean plate 3 times.In all remaining cleaning steps, cleaning buffer solution keeps identical, and During third time is cleaned, before cleaning solution is removed completely, plate is incubated at room temperature 10 minutes.Add with including 0.1% 10mM PBS pH 7.4,150mM NaCl, the 1mM CaCl of Tween-20₂、0.5mM MgCl₂In 3%BSA with 1:10000 times The anti-rotavirus rabbit polyclonal antibody (being provided by Koki Taniguchi professor's good wills) diluted, and by plate in 37 DEG C of incubations 1 hour.Then, plate is cleaned 3 times, and added in the 10mM PBS pH 7.4 comprising 0.1% Tween-20,150mM NaCl, 1mM CaCl₂In 3%BSA in 1:Goat anti-rabbit antibodies (the 111- of 5000 times of horseradish peroxidases-conjugated diluted 035-144, Jackson Immunoresearch, West Grove, PA), and plate is incubated 1 hour in 37 DEG C.Plate is cleaned 3 It is secondary.After last cleaning, by plate and SureBlue TMB peroxidase substrates (KPL, Gaithersburg, MD) in room Temperature is lower to be incubated 20 minutes.By adding 1N HCl come terminating reaction, and use Multiskan Ascent ELIASAs (Thermo Scientific, Waltham, MA) measurement A450 values.

The generation of class rotaviral particles comprising VP2 and VP6

By in Ben Saimushi tobaccos (Nicotiana benthamiana) transient expression generate comprising VP2 and VP6 Class rotaviral particles.With including the 1 of AGL1/1710 and AGL1/1713:The Agrobacterium inoculation thing of 1 mixture enters to plant Row agroinfiltration, and harvesting preincubation 7 days.It is pure from biomass using the method described in material and method part Dissolve class rotaviral particles.After the extract of clarification is centrifuged in Iodixanol density gradient, by being contaminated through coomassie The SDS-PAGE of color, preceding ten fractions started at from bottom of the tube are analyzed.As shown in Figure 45 A, wheel virus antigen (VP2 and VP6) is mainly found in the fraction 2 and fraction 3 of density gradient, and the concentration of wherein Iodixanol is about 35%, The concentration lower class rotaviral particles are expected to be found.The phytoprotein pollution found in these fractions is few.With anti- The Western blot analysis that rotavirus hyperimmune rabbit anteserum and polyclonal rabbit-anti VP2 antibody are carried out to fraction confirms density VP2 and VP6 identity (Figure 45 B and 45C) in gradient fractions.Fraction 2 is merged with fraction 3, passes through high speed centrifugation and settling flux Iodixanol is removed, the particle of purifying is delivered into cryo EM analysis (NanoImaging Services Inc., La Jolla, CA), the particles of similar rotaviral particles is assembled into confirm VP2 with VP6.(left part) as shown in figure 49, Cryo EM (cryoEM) image confirmings of VP2/VP6 particles antigen is correctly assembled into class rotaviral particles.

The generation of class rotaviral particles comprising VP2, VP6 and VP7

By in Ben Saimushi tobaccos (Nicotiana benthamiana) transient expression generate comprising VP2, VP6 With VP7 class rotaviral particles.With 1 comprising AGL1/1710, AGL1/1713, AGL1/1734:1:The agriculture bar of 1 mixture Bacterium inoculum is harvesting preincubation 7 days to plant agroinfiltration.Using the method described in material and method part, Class rotaviral particles are purified into from biomass.After the extract of clarification is centrifuged in Iodixanol density gradient, make With the SDS-PAGE dyed through coomassie, preceding ten fractions started at from bottom of the tube are analyzed.As shown in Figure 46 A, Wheel virus antigen (VP2, VP6 and VP7) is mainly found in the fraction 2 of density gradient with fraction 3, wherein Iodixanol Concentration is about 35%, is expected to be found in the concentration lower class rotaviral particles.The vegetable protein found in these fractions Matter pollution is few.The western blot carried out with anti-rotavirus hyperimmune rabbit anteserum and polyclonal rabbit-anti VP7 antibody to fraction It has been analyzed to identify the identity (Figure 46 B and 46C) of VP6 and VP7 in density gradient fraction.

The generation of class rotaviral particles comprising VP2, VP4, VP6 and VP7

By in Ben Saimushi tobaccos (Nicotiana benthamiana) transient expression generate comprising VP2, VP4, VP6 and VP7 class rotaviral particles.With 1 comprising AGL1/1710, AGL1/1730, AGL1/1713, AGL1/1734:1: 1:The Agrobacterium inoculation thing of 1 mixture has carried out agroinfiltration to plant, and is harvesting preincubation 7 days.Using material and Method described in method part, class rotaviral particles are purified into from biomass.By the extract of clarification in iodine gram sand After being centrifuged in alcohol density gradient, preceding ten fractions started at from bottom of the tube are carried out using through the SDS-PAGE that coomassie dyes Analysis.As shown in Figure 47 A, 3 kinds (VP2, VP6 and VP7) in 4 kinds of wheel virus antigens can be observed, their main quilts It is found in the fraction 3 of density gradient, the concentration of wherein Iodixanol is about 35%, pre- in the concentration lower class rotaviral particles Phase will be found.The phytoprotein pollution found in these fractions is few.It is contemplated that in the gel through coomassie dyeing not The VP4 of detectable level be present, because can not when carrying out same analysis on purified human rotavirus particle It was observed that VP4.Western blot is carried out to fraction with anti-rotavirus hyperimmune rabbit anteserum and polyclonal rabbit-anti VP7 antibody It has been analyzed to identify the identity (Figure 47 B and 47C) of VP6 and VP7 in density gradient fraction.Level is eliminated by high speed centrifugation and resuspension The Iodixanol divided in 3, and purified particle is analyzed by ELISA, to confirm VP4 presence.In Figure 48 Shown result is clearly illustrated, background signal water is generated because the negative control particle comprising VP2/VP6 and VP7 only results in It is flat, therefore ELISA specific recognitions VP4.On the contrary, when testing under the same conditions, to comprising VP2, VP4, VP6 and The analysis of the purified particle of 3 different batches of VP7 antigens shows strong and uniform signal.By purified VP2/ VP4/VP6/VP7RLP delivers to cryo EM analysis (NanoImaging Services Inc., La Jolla, CA), To confirm that four kinds of antigens are assembled into the particle of similar rotaviral particles.(right part) as shown in figure 49, VP2/VP4/ Cryo EM (cryoEM) image confirmings of VP6/VP7 particles antigen is correctly assembled into class rotaviral particles.

Table 6 lists the sequence provided in multiple embodiments of the present invention.

Table 6：The sequence explanation of sequence identifier

Bibliography

Yang Y M,Li X,Yang H,et al.Immunogenicity and virus-like particle formation of rotavirus capsid produced in transgenic plants.Sci China Life Sci,2011,54:82-89

Angel,J.,Franco,M.A.and Greenberg,H.B.(2007).Rotavirus vaccines: recent developments and future considerations.Nature reviews:Microbiology 5, 529-539

Araujo,I.T.,Ferreira,M.S.R.and Failho,A.M.(2001).Rotavirus genotypes [4]G9,P[6]G9,and P[8]G9in hospitalized children with acute gastroenteritis in Rio de Janeiro,Brazil.J Clin Microbiol 391999–2001.

Arias,C.F.,Ballado,T.and Plebafiski,M.(1986).Synthesis of the outer- capsid glycoprotein of the simian rotavirus SA11in Escherichia coli.Gene 47, 211-219

Au K.S.,Mattion N.M.,Estes M.K.,(1993).A Subviral Particle Binding Domain on the Rotavirus Nonstructural Glycoprotein NS28.Virology 194,665-67

Balen B,Krsnik-Rasol M,(2007).N-glycosylation of recombinant therapeutic glycoproteins in plant systems.Food Technology and Biotechnology 451-10.

Bardor,M.,Faveeuw,C.,Fitchette,A.C.,Gilbert,D.,Galas,L.,Trottein,F., Faye,L.and Lerouge P.(2003).Immunoreactivity in mammals of two typical plant glycoepitopes,core alpha(1,3)-fucose and core xylose.Glycobiology 13427-434

Berois,M.,Sapin,C.,Erk,I.,Poncet,D.and Cohen,J.(2003).Rotavirus Nonstructural Protein NSP5Interacts with Major Core Protein VP2.Journal of virology 77,1757

Bertolotti-Ciarlet,A.,Ciarlet,M.,Crawford,S.E.,Conner,M.E.and Estes, M.K.(2003).Immunogenicity and protective efficacy of rotavirus 2/6-virus-like particles produced by a dual baculovirus expression vector and administered intramuscularly,intranasally,or orally to mice.Vaccine 21,3885-3900

Chen,J.Z.,Settembre,E.C.,Aoki,A.T.,Zhang,X.,Bellamy,A.R.,Dormitzer, P.R.,Harrison,S.C.and Grigorieff,N.(2009).Molecular interactions in rotavirus assembly and uncoating seen by high-resolution cryo-EM.PNAS 106,10644-10648

Crawford,S.E.,Estes,M.K.,Ciarlet,M.,Barone,C.,O'Neal,C.M.,Cohen,J.and Conner,M.E.(1999).Heterotypic protection and induction of a broad heterotypic neutralization response by rotavirus-like particles.Journal of Virology 73, 4813-4822

Crawford,S.E.,Labbe,M.,Cohen,J.,Burroughs,M.H.,Zhou,Y.J.and Estes, M.K.(1994).Characterization of virus-like particles produced by the expression of rotavirus capsid proteins in insect cells.Journal of virology 68,5945-5952

Denisova,E.R.,Dowling,W.,LaMonica,R.,Shaw,R.,Scarlata,S.,Ruggeri, F.and Mackow,E.R.(1999).Rotavirus Capsid Protein VP5*Permeabilizes Membranes.Journal of Virology 733147-3153

Dennehy,P.H.(2007).Rotavirus vaccines-An update.Vaccine 25,3137-3141

Estes M.K(1996).Rotavirus and their replication.Fields Virology 2, 1625-1655Fabbretti,E.,Afrikanova,I.,Vascotto,F.and Burrone,O.R.(1999).Two nonstructural rotavirus proteins,NSP2and NSP5,form viroplasm-like structures in vivo.J Gen Virol 80333-9.

Favacho,A.R.,Kurtenbach,E.,Sardi,S.I.and Gouvea,V.S.(2006).Cloning, expression,and purification of recombinant bovine rotavirus hemagglutinin, VP8*,in Escherichia coli.Protein Expression and Purification 46,196-203

Gentsch,J.R.,Laird,A.R.,Bielfelt,B.,Griffin,D.D.,Bányai,K., Ramachandran,M.,Jain,V.,Cunliffe,N.A.,Nakagomi,O.,Kirkwood,C.D.,Fischer,T.K., Parashar,U.D.,Bresee,J.S.,Jiang,B.and Glass,R.I.(2005).Serotype Diversity and Reassortment between Human and Animal Rotavirus Strains:Implications for Rotavirus Vaccine Programs J Infect Dis.192,S146-59

Glass,R.I.,Parashar,U.D.,Bresee,J.S.,Turcios,R.,Fischer,T.K., Widdowson,MA.,Jiang,B.amd Gentsch,J.R.(2006).Rotavirus vaccines:current prospects and future challenges.Lancet 368,323–32

González,A.M.,Nguyen,T.V.,Azevedo,M.S.P.,Jeong,K.,Agarib,F.,Iosef,C., Chang,K.,Lovgren-Bengtsson,K.,Morein,B.and Saif,L.J.(2004).Antibody responses to human rotavirus(HRV)in gnotobiotic pigs following a new prime/boost vaccine strategy using oral attenuated HRV priming and intranasal VP2/ 6rotavirus-like particle(VLP)boosting with ISCOM.Clinical&Experimental Immunology 135361–372

González,R.A.,Espinosa,R.,Romero,P.,López,S.and Arias C.F.(2000) .Relative localization of viroplasmic and endoplasmic reticulum-resident rotavirus proteins in infected cells.Archives of Virology 145,1963–1973

Greenberg,H.B.and Estes,M.K.(2009).Rotaviruses:From Pathogenesis to Vaccination.Gastroenterology 136,1939-1951

Hoshino,Y.,Jones,R.W.and Kapikian,A.Z.(1998).Serotypic characterization of outer capsid spike protein VP4of vervet monkey rotavirus SA11strain.Archives of Virology 143,1233-1244

Istrate,C.,Hinkula,J.,Charpilienne,A.,Poncet,D.,Cohen,J.,Svensson, L.and Johansen,K.(2008).Parenteral administration of RF 8-2/6/7rotavirus-like particles in a one-dose regimen induce protective immunity in mice.Vaccine 26,4594-4601

Khodabandehloo,M.,Shamsi,S.M.,Shahrabadi,Keyvani,H.and Bambai,B. (2009).Cloning and Expression of Simian Rotavirus Spike Protein(VP4)in Insect Cells by Baculovirus Expression System.Iranian Biomedical Journal 139-18

Kim,Y.,Nielsen,P.R.,Hodgins,D.,Chang,K.O.and Saif,L.J.(2002) .Lactogenic antibody responses in cows vaccinated with recombinant bovine rotavirus-like particles(VLPs)of two serotypes or inactivated bovine rotavirus vaccines.Vaccine 20,1248–1258

Kovacs-Nolan J.,Erika Sasaki,Dongwan Yoo,Yoshinori Mine,Cloning and Expression of Human Rotavirus Spike Protein,VP8*,in Escherichia coli.Biochemical and Biophysical Research Communications,282,1183-1188

Lawton,J.A.,Estes,M.K.and Venkataram,P.B.V.(2000).Mechanism of genome transcription in segmented dsRNA viruses.Advances in Virus Research 55,185- 214

Lopez,T.,Camacho M.,Zayas M.,Najera R.,Sanchez R.,Arias C.F.and Lopez S.(2005).Silencing the Morphogenesis of Rotavirus.Journal of Virology 79,184– 92

Lundgren,O.and Svensson,L.(2001).Pathogenesis of Rotavirus diarrhoea.Microbes and Infection 31145-1156

Madore,H.P.,Estes,M.K.,Zarley,C.D.,Hu,B.,Parsons,S.,Digravio,D., Greiner,S.,Smith,R.,Jiang,B.,Corsaro,B.,Barniak,V.,Crawford,S.and Conner,M.E. (1999).Biochemical and immunologic comparison of virus-like particles for a rotavirus subunit vaccine.Vaccine 17,2461-2471

Marusic,C.,Rizza,P.,Lattanzi,L.,Mancini,C.,Spada,M.,Belardelli,F., Benvenuto,E.and Capone,I.(2001).Chimeric plant virus particles as immunogens for inducing murine and human immune responses against human immunodeficiency virus type 1.Journal of Virology 75,8434–8439.

Matsumura,T.,Itchoda,N.and Tsunemitsu,H.(2002).Production of immunogenic VP6protein of bovine group A rotavirus in transgenic potato plants.Archives of Virology 147,1263-1270

Mena,J.A.,Ramírez,O.T.and Palomares,L.A.(2006).Intracellular distribution of rotavirus structural proteins and virus-like particles expressed in the insect cellbaculovirus system.Journal of Biotechnology 122, 443-452

Meyer,J.C.,Bergmann,C.C.and Bellamy,A.R.(1989).Interaction of rotavirus cores with the nonstructural glycoprotein NS28.Virology 171,98-107

Molinari,P.,Peralta,A.and Taboga,O.(2008).Production of rotavirus- like particles in Spodoptera frugiperda larvae.Journal of Virological Methods 147,364-367

Nilsson,M.,von Bonsdorff,C.H.,Weclewicz,K.,Cohen,J.and Svensson,L. (1998).Assembly of viroplasm and virus-like particles of rotavirus by a Semliki Forest virus replicon.Virology 242,255-265

Nishikawa,K.,Fukuhara,N.,Liprandi,F.,Green,K.,Kapikian,A.,Chanock, R.and Gorziglia,M.(1989).VP4protein of porcine rotavirus strain OSU expressed by a baculovirus recombinant induces neutralizing antibodies.Virology 173, 631-637

O'Brien,G.J.,Bryant,C.J.,Voogd,C.,Greenberg,H.B.,Gardner,R.C.and Bellamy,A.R.(2000).Rotavirus VP6expressed by PVX vectors in Nicotiana benthamiana coats PVX rods and also assembles into virus-like particles.Virology 270,444-453

Palombo,E.A.(1999).Genetic and antigenic diversity of human rotaviruses:potential impact on the success of candidate vaccines.FEMS Microbiology Letters 181,1-8

Palomares,L.A.and Ramirez,O.T.(2009).Challenges for the production of virus-like particles in insect cells:The case of rotavirus-like particles.BiochemicalEngineering Journal 45(3),158-167

Peralta,A.,Molinari,P.and Taboga,O.(2009).Chimeric recombinant rotavirus-like particles as a vehicle for the display of heterologous epitopes.Virology Journal 6,192

Ramachandran,M.,Kirkwood,C.D.,Unicomb,L.,Cunliffe,N.A.,Ward,R.L., Bhan,M.K.,Clark,H.F.,Glass,R.I.and Gentsch,J.R.(2000).Molecular characterization of serotype G9rotavirus strains from a global collection.Virology 278,436-444

Ribes,J.M.,Ortego,J.,Ceriani,J.,Montava,R.,Enjuanes,L.and Buesa,J. (2011).Transmissible gastroenteritis virus(TGEV)-based vectors with engineered murine tropism express the rotavirus VP7 protein and immunize mice against rotavirus.Virology 410107-118

Rodríguez-Limas,W.A.,Tyo,K.E.J.,Nielsen,J.,Ramírez,O.T.and Palomares, L.A.(2011).Molecular and process design for rotavirus-like particle production in Saccharomyces cerevisiae.Microb Cell Fact.10,33

Saldana,S.,Esquivel Guadarrama,F.,Olivera Flores Tde,J.,Arias,N., Lopez,S.,Arias,C.,Ruiz-Medrano,R.,Mason,H.,Mor,T.,Richter,L.,Arntzen,C.J.and Gomez Lim,M.A.(2006).Production of rotavirus-like particles in tomato (Lycopersicon esculentum L.)fruit by expression of capsid proteins VP2and VP6and immunological studies.Viral Immunology 19,42-53

Sanchez-Padilla,E.,Grais,R.F.,Guerin,P.J.,Steele,A.D.,Burny,M.E.and Luquero,F.J.(2009).Burden of disease and circulating serotypes of rotavirus infection in sub-Saharan Africa:systematic review and meta-analysis.Lancet Infectious Diseases 9,567-576.

Steele,A.D.,Ivanoff,B.and African Rotavirus Network(2003).Rotavirus strains circulating in Africa during 1996-1999:emergence of G9strains and P [6]strains.Vaccine 21,361-367

Tian,P.,Ball,J.M.,Zeng,C.Q.Y.and Estes,M.K.(1996).The Rotavirus Nonstructural Glycoprotein NSP4Possesses Membrane Destabilization Activity.Journal of Vriology 70,6973–6981

Thongprachum,A.,Chaimongkol,N.,Khamrin,P.,Pantip,C.,Mizuguchi,M., Ushijima,H.and Maneekarn,N.(2010).A novel multiplex RT-PCR for identification of VP6subgroups of human and porcine rotaviruses,Journal of Virological Methods,168,191-196

Trabelsi,A.,Peenze,I.,Pager,C.,Jeddi,M.and Steele,D.(2000) .Distribution of Rotavirus VP7Serotypes and VP4Genotypes Circulating in Sousse,Tunisia,from 1995to 1999:Emergence of Natural Human Reassortants.Journal of Clinical Microbiology 38,3415-3419

Varani,G.and Allain,F.H-T.(2002).How a rotavirus hijacks the human protein synthesis machinery.Nature Structural Biology 9,158–160.

Vende,P.,Taraporewala,Z.F.and Patton,J.T.(2002).RNA-Binding Activity of the Rotavirus Phosphoprotein NSP5Includes Affinity for Double-Stranded RNA.Journal of Virology 76,5291-5299.

Vesikari,T.,Karvonen,A.,Korhonen,T.,Espo,M.,Lebacq,E.,Forster,J., Zepp,F.,Delem,A.and De Vos,B.(2004).Safety and immunogenicity of RIX4414live attenuated human rotavirus vaccine in adults,toddlers and previously uninfected infants.Vaccine 22,2836-2842

Vézina,L.-P.,Faye,L.,Lerouge,P.,D’Aoust,M.-A.,Marquet-Blouin,E., Burel,C.,Lavoie,P.-O.,Bardor,M.and Gomord,V.(2009),Transient co-expression for fast and high-yield production of antibodies with human-like N-glycans in plants.Plant Biotechnology Journal 7,442-455.

Zhou,B.,Zhang,Y.,Wang,X.,Dong,J.,Wang,B.,Han,C.,Yu,J.,Li,D.(2010) .Oral administration of plant-based rotavirus VP6induces antigen-specific IgAs,IgGs and passive protection in mice.Vaccine 28, 6021-6027

Zhou,H.,Guo,L.,Wang,M.,Qu,J.,Zhao,Z.,Wang,J.and Hung,T.(2011).Prime immunization with rotavirus VLP 2/6followed by boosting with an adenovirus expressing VP6induces protective immunization against rotavirus in mice.Virology Journal 8,3

All references document is both incorporated herein by reference.

The present invention is described on one or more embodiments.However, it will be clear to a person skilled in the art that In the case of without departing substantially from the scope of the present invention as defined in the claims, substantial amounts of changes and modifications can be carried out.

Claims (28)

1. the method for class rotaviral particles (RLP), methods described bag are produced in plant, the part of plant or plant cell Include：

A) following first nucleic acid, following second nucleic acid and following 3rd nucleic acid are introduced, first nucleic acid is included in the plant In it is active and be operatively connectable to encode the of the first nucleotide sequence of the first colyliform viral structural proteinses matter One control region, the first colyliform viral structural proteinses matter are selected from VP2, VP6 and VP7, and second nucleic acid is included in described The second nucleotide sequence that is active and being operatively connectable to encode the second rotavirus structural protein matter in plant The second control region, the second rotavirus structural protein matter is selected from VP2, VP6 and VP7, and the 3rd nucleic acid is included in Trinucleotide that is active and being operatively connectable to encode third round shape viral structural proteinses matter in the plant 3rd control region of sequence, the third round shape viral structural proteinses matter are selected from VP2, VP6 and VP7, wherein, VP2, VP6, And every kind of in VP7 is introduced into the plant, the part of plant or plant cell,

Wherein, first, second or third nucleotide sequence for encoding VP7 includes the signal peptide truncated, and wherein VP7's is natural 1-29 amino acids in signal peptide are lacked；

B) cultivated under conditions of the first, second, and third nucleic acid transient expression is allowed the plant, plant part or Plant cell, the RLP is thus produced,

C) plant, the part of plant or plant cell are harvested, and

D) it is being Ca comprising the calcium ion no more than 10mM²⁺Buffer solution in the presence of from the plant, the part of plant or plant RLP described in thing cell extraction, also, be Ca comprising the calcium ion no more than 10mM²⁺Buffer solution in the presence of purifying described in RLP。

2. according to the method for claim 1, wherein following 4th nucleic acid are introduced into step a), also, when in step It is expressed when the plant, the part of plant or plant cell are cultivated in b), the 4th nucleic acid, which is included in the plant, to be had Active and being operatively connectable to encode the tetranucleotide sequence of fourth round shape viral structural proteinses matter the 4th adjusts Area is controlled, the fourth round shape viral structural proteinses matter is VP4.

3. according to the method for claim 1, wherein, first, second, and third nucleic acid is with 1:1:1 ratio is introduced into The plant, the part of plant or plant cell.

4. according to the method for claim 2, wherein, the first, second, third and fourth nucleic acid is with 1:1:1:1 ratio Example is introduced into the plant, the part of plant or plant cell.

5. method according to claim 1 or 2, wherein, the codon of the nucleotide sequence uses is directed to people's password Son uses, the G/C content of raising or its combination are optimized.

6. according to the method for claim 1, wherein, first, second, and third nucleotide sequence is operationally connected It is connected to transcription enhancer element.

7. according to the method for claim 6, wherein, the transcription enhancer element is adjusted comprising cowpea mosaic virus (CPMV) Control area.

8. according to the method for claim 1, wherein first, second or third nucleotide sequence or combinations thereof quilt It is operatively connectable to cowpea mosaic virus (CPMV) control region.

9. according to the method for claim 2, wherein described first, second, third or tetranucleotide sequence or they Combination is operatively connected to cowpea mosaic virus (CPMV) control region.

10. according to the method for claim 1, wherein it is described first, second or third nucleotide sequence coded, include or Encode and target sequence comprising one or more of compartments.

11. according to the method for claim 10, wherein the compartment targets sequence by one or more colyliform disease Malicious structural protein matter guides endoplasmic reticulum (ER), chloroplaset, plastid or the apoplast of the plant cell into.

12. according to the method for claim 11, wherein compartment targeting sequential coding apoplast signal peptide or plastid letter Number peptide.

13. by RLP caused by the method according to claim 11, wherein the RLP contains plant comprising one or more The rotavirus structural protein matter of thing specificity N- glycan or N- glycan through modification.

14. composition, the composition include effective dose be used to induce immune response in subject will according to right Ask the RLP described in 13, and pharmaceutical acceptable carrier.

15. according to the method for claim 1, wherein, the nucleotide sequence for encoding VP2 is included by SEQ ID NO：13、SEQ ID NO：14 or SEQ ID NO：Nucleotide sequence defined in 45, the nucleotide sequence for encoding VP6 are included by SEQ ID NO： 17、SEQ ID NO：18 or SEQ ID NO：Nucleotide sequence defined in 46, the nucleotide sequence for encoding VP7 are included by SEQ ID NO：19th, nucleotide sequence defined in 20,48,49,52,53,54 or 57.

16. according to the method for claim 2, wherein, the nucleotide sequence for encoding VP2 is included by SEQ ID NO：13、SEQ ID NO：14 or SEQ ID NO：Nucleotide sequence defined in 45, the nucleotide sequence for encoding VP6 are included by SEQ ID NO： 17、SEQ ID NO：18 or SEQ ID NO：Nucleotide sequence defined in 46, the nucleotide sequence for encoding VP7 are included by SEQ ID NO：19th, nucleotide sequence defined in 20,48,49,52,53,54 or 57, VP4 are included by SEQ ID NO：15、16、47、 Nucleotide sequence defined in 50 or 51.

17. the method for class rotaviral particles (RLP), methods described bag are produced in plant, the part of plant or plant cell Include：

A) following plants, the part of plant or plant cell are provided, the plant, the part of plant or plant cell include following First nucleic acid, following second nucleic acid and following 3rd nucleic acid, first nucleic acid be included in the plant in it is active and It is operatively connectable to encode the first control region of the first nucleotide sequence of the first colyliform viral structural proteinses matter, described the One rotavirus structural protein matter is selected from VP2, VP6 and VP7, and second nucleic acid is included in active in the plant And it is operatively connectable to encode the second control region of the second nucleotide sequence of the second rotavirus structural protein matter, institute State the second rotavirus structural protein matter and be selected from VP2, VP6 and VP7, the 3rd nucleic acid, which includes, to be included in the plant The 3rd of trinucleotide sequence that is active and being operatively connectable to coding third round shape viral structural proteinses matter Control region, the third round shape viral structural proteinses matter are selected from VP2, VP6 and VP7, wherein, the plant, the part of plant Or plant cell is comprising every kind of in VP2, VP6 and VP7；

Also, first, second or third nucleotide sequence for wherein, encoding VP7 includes the signal peptide truncated, wherein VP7 Natural signals peptide in 1-29 amino acids lacked；

B) cultivated under conditions of the first, second, and third nucleic acid transient expression is allowed the plant, plant part or Plant cell, the RLP is thus produced,

C) plant, the part of plant or plant cell are harvested, and

D) it is being Ca comprising the calcium ion no more than 10mM²⁺Buffer solution in the presence of from the plant, the part of plant or plant RLP described in thing cell extraction, also, be Ca comprising the calcium ion no more than 10mM²⁺Buffer solution in the presence of purifying described in RLP。

18. according to the method for claim 17, wherein to the plant, the part of plant or plant cell in step a) There is provided following 4th nucleic acid, the 4th nucleic acid is included in active in the plant and is operatively connectable to coding the 4th control region of the tetranucleotide sequence of four colyliform viral structural proteinses matter, also, when described in the cultivation in step b) The fourth round shape viral structural proteinses matter is expressed when plant, the part of plant or plant cell, wherein, the fourth round Shape viral structural proteinses matter is VP4.

19. the method according to claim 17 or 18, wherein, the codon of the nucleotide sequence is close using people is directed to Numeral using, improve G/C content or its combination be optimized.

20. according to the method for claim 17, wherein, first, second, and third nucleotide sequence is by operationally It is connected to transcription enhancer element.

21. according to the method for claim 20, wherein, the transcription enhancer element includes cowpea mosaic virus (CPMV) Control region.

22. according to the method for claim 17, wherein it is described first, second or third nucleotide sequence coded, include or Encode and target sequence comprising one or more of compartments.

23. according to the method for claim 22, wherein the compartment targets sequence by one or more colyliform disease Malicious structural protein matter guides endoplasmic reticulum (ER), chloroplaset, plastid or the apoplast of the plant cell into.

24. according to the method for claim 22, wherein compartment targeting sequential coding apoplast signal peptide or plastid letter Number peptide.

25. by RLP caused by the method according to claim 11, wherein the RLP include it is one or more containing The rotavirus structural protein matter of plant specificity N- glycan or N- glycan through modification.

26. composition, the composition include effective dose be used to induce immune response in subject will according to right Ask the RLP described in 25, and pharmaceutical acceptable carrier.

It is used to induce the immunity to rotavirus infection in subject 27. the composition described in claim 26 is used to manufacture Medicament purposes.

It is used to induce the immunity to rotavirus infection in subject 28. the composition described in claim 14 is used to manufacture Medicament purposes.